Anti-aging compositions and methods for using same

ABSTRACT

The invention generally relates to anti-aging compositions and methods for using the anti-aging compositions. In one aspect, the invention provides a method for preventing cellular aging in a cell of a subject, the method comprising the step of providing to the cell an effective amount of at least one lithium compound or a pharmaceutically acceptable salt thereof, and an effective amount of at least one glycyrrhizie triterpenoid compound or a pharmaceutically acceptable salt thereof, thereby preventing the cell from aging. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Application No. 15/773,967filed May 4, 2018, which claims priority to PCT Application No.PCT/US2016/060371 filed Nov. 3, 2016, which claims the benefit ofpriority to U.S. Provisional Application No. 62/250,910 filed Nov. 4,2015, which are 15/incorporated herein by reference in its entirety.

FIELD OF INVENTION

This disclosure relates to anti-aging compositions and methods for usingsame.

BACKGROUND

Cellular aging processes are important and have been implicated, inpart, in many age-related diseases or conditions among aged people.There remains a lack of effective prevention or treatment methods forage-related diseases or disorders. In view of the increasing populationof aged people, the rising cost of health care associated with theirtreatment, there remains a need for effective anti-aging compositionsfor the prevention or treatment of age-related diseases or disorders.This need and other needs are satisfied by the various aspects of thepresent disclosure.

SUMMARY

In accordance with the purpose(s) of the invention, as embodied andbroadly described herein, the invention, in one aspect, relates toanti-aging methods using known compounds and pharmaceutical compositionscomprising same.

Disclosed are methods for preventing cellular aging in a cell of asubject, the method comprising the step of providing to the cell aneffective amount of at least one lithium compound or a pharmaceuticallyacceptable salt thereof, and an effective amount of at least oneglycyrrhizie triterpenoid compound or a pharmaceutically acceptable saltthereof, thereby preventing the cell from aging.

Also disclosed are methods for preventing cellular aging activity in asubject, the method comprising the step of providing to the subject aneffective amount of at least one intracellular alkalinizing agent or apharmaceutically acceptable salt thereof, thereby preventing thecellular aging activity.

Also disclosed are methods method for the treatment of a subject, themethod comprising the steps of: diagnosing the subject as having anage-related disorder or disease; and administering to the subject aneffective amount of at least one lithium compound or a pharmaceuticallyacceptable salt thereof, and an effective amount of at least oneglycyrrhizie triterpenoid compound or a pharmaceutically acceptable saltthereof.

Also disclosed are pharmaceutical and nutraceutical compositionscomprising an effective amount of at least one lithium compound or apharmaceutically acceptable salt thereof and an effective amount of atleast one glycyrrhizie triterpenoid compound or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier.

Also disclosed are kits comprising an effective amount of at least onelithium compound or a pharmaceutically acceptable salt thereof, aneffective amount of at least one glycyrrhizie triterpenoid compound or apharmaceutically acceptable salt thereof, and one or more of: a) atleast one agent known to treat an age-related disorder or disease; b)instructions for treating the age-related disorder or disease; and c)instructions for administering the lithium compound and the glycyrrhizietriterpenoid compound in connection with the age-related disorder ordisease.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1 show lithium and glycyrrhizic acid combination treated fibroblastsamples stained for beta-galactosidase.

FIG. 2 show glycyrrhizic acid monotherapy treated fibroblast samplesstained for beta-galactosidase.

FIG. 3 show lithium monotherapy treated fibroblast samples stained forbeta-galactosidase.

FIG. 4 show untreated treated fibroblast samples stained forbeta-galactosidase.

FIG. 5 show representative data pertaining to fibroblast samples.

FIG. 6 show untreated treated fibroblast samples stained forbeta-galactosidase.

FIG. 7 show glycyrrhizic acid monotherapy treated fibroblast samplesstained for beta-galactosidase.

FIG. 8 show lithium monotherapy treated fibroblast samples stained forbeta-galactosidase.

FIG. 9 show lithium monotherapy treated fibroblast samples stained forbeta-galactosidase.

FIG. 10 show lithium monotherapy treated fibroblast samples stained forbeta-galactosidase.

FIG. 11 show lithium monotherapy treated fibroblast samples stained forbeta-galactosidase.

FIG. 12 show lithium and glycyrrhizic acid combination treatedfibroblast samples stained for beta-galactosidase.

FIG. 13 show lithium and glycyrrhizic acid combination treatedfibroblast samples stained for beta-galactosidase.

FIG. 14 show lithium and glycyrrhizic acid combination treatedfibroblast samples stained for beta-galactosidase.

FIG. 15 show lithium and glycyrrhizic acid combination treatedfibroblast samples stained for beta-galactosidase.

FIG. 16 show representative data pertaining gene expression profile ofthe fibroblast in control and treatments groups.

FIG. 17 show representative data pertaining survival profile of C.elegans specimens in control and treatments groups.

FIG. 18 show untreated treated fibroblast samples stained forbeta-galactosidase.

FIG. 19 show retinoic acid monotherapy treated fibroblast samplesstained for beta-galactosidase.

FIG. 20 show lithium, glycyrrhizic acid, and retinoic acid combinationtreated fibroblast samples stained for beta-galactosidase.

FIG. 21 show lithium and glycyrrhizic acid combination treatedfibroblast samples stained for beta-galactosidase.

FIG. 22 shows a graph showing blood telomere related gene expressiondata following oral administration of lithium and glycyrrhizic acidcombination.

FIG. 23 shows a heatmap showing the effect of inhaled lithium ontelomere lengthening.

FIG. 24 shows a heatmap showing results of the effect of lithium andglycyrrhizic acid combination on gene expression of various biomarkers.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon. Nothing herein is tobe construed as an admission that the present invention is not entitledto antedate such publication by virtue of prior invention. Further, thedates of publication provided herein may be different from the actualpublication dates, which can require independent confirmation.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “subject” can be a vertebrate, such as amammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject ofthe herein disclosed methods can be a human, non-human primate, horse,pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In one aspect, the subject is a mammal. A patient refers to asubject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects. In some aspects of the disclosedmethods, the subject has been diagnosed with a need for treatment of oneor more disorders prior to the administering step. In some aspects ofthe disclosed methods, the subject has been diagnosed with a need fortreatment of one or more age-related disorder or disease prior to theadministering step. In some aspects of the disclosed method, the subjecthas been diagnosed with a chronic pulmonary disease prior to theadministering step. In some aspects of the disclosed method, the subjectbeen diagnosed with a chronic pulmonary disease prior to theadministering step.

As used herein, the term “treatment” or “treating” refers to the medicalmanagement of a patient with the intent to cure, ameliorate, stabilize,or prevent a disease, pathological condition, or disorder. This termincludes active treatment, that is, treatment directed specificallytoward the improvement of a disease, pathological condition, ordisorder, and also includes causal treatment, that is, treatmentdirected toward removal of the cause of the associated disease,pathological condition, or disorder. In addition, this term includespalliative treatment, that is, treatment designed for the relief ofsymptoms rather than the curing of the disease, pathological condition,or disorder; preventative treatment, that is, treatment directed tominimizing or partially or completely inhibiting the development of theassociated disease, pathological condition, or disorder; and supportivetreatment, that is, treatment employed to supplement another specifictherapy directed toward the improvement of the associated disease,pathological condition, or disorder. In various aspects, the term coversany treatment of a subject, including a mammal (e.g., a human), andincludes: (i) preventing the disease from occurring in a subject thatcan be predisposed to the disease but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, i.e., arresting its development;or (iii) relieving the disease, i.e., causing regression of the disease.In one aspect, the subject is a mammal such as a primate, and, in afurther aspect, the subject is a human. The term “subject” also includesdomesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle,horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse,rabbit, rat, guinea pig, fruit fly, etc.).

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed. For example, preventing age-related disease or disorder meansreducing the incidences, delaying or reversing diseases or disordersthat are related to or associated with aging.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein. In some aspects ofthe disclosed methods, the subject has been diagnosed with a need fortreatment of an age-related disorder or disease prior to theadministering step. As used herein, the phrase “identified to be in needof treatment for a disorder,” or the like, refers to selection of asubject based upon need for treatment of the disorder. It iscontemplated that the identification can, in one aspect, be performed bya person different from the person making the diagnosis. It is alsocontemplated, in a further aspect, that the administration can beperformed by one who subsequently performed the administration.

As used herein, the term “providing” refers to any method ofadministering or contacting a disclosed compound or composition to acell, target receptor, or other biological entity. preparation to asubject. Such methods are well known to those skilled in the art andinclude, but are not limited to, oral administration, transdermaladministration, administration by inhalation, nasal administration,topical administration, intravaginal administration, ophthalmicadministration, intraaural administration, intracerebral administration,rectal administration, and parenteral administration, includinginjectable such as intravenous administration, intra-arterialadministration, intramuscular administration, and subcutaneousadministration. Administration can be continuous or intermittent. Invarious aspects, a preparation can be administered therapeutically; thatis, administered to treat an existing disease or condition. In furthervarious aspects, a preparation can be administered prophylactically;that is, administered for prevention of a disease or condition.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, and parenteral administration, including injectable suchas intravenous administration, intra-arterial administration,intramuscular administration, and subcutaneous administration.Administration can be continuous or intermittent. In various aspects, apreparation can be administered therapeutically; that is, administeredto treat an existing disease or condition. In further various aspects, apreparation can be administered prophylactically; that is, administeredfor prevention of a disease or condition.

The term “contacting” as used herein refers to bringing a disclosedcompound and a cell, target receptor, or other biological entitytogether in such a manner that the compound can affect the activity ofthe target (e.g., receptor, cell, etc.), either directly; i.e., byinteracting with the target itself, or indirectly; i.e., by interactingwith another molecule, co-factor, factor, or protein on which theactivity of the target is dependent.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, a“therapeutically effective amount” refers to an amount that issufficient to achieve the desired therapeutic result or to have aneffect on undesired symptoms, but is generally insufficient to causeadverse side effects. The specific therapeutically effective dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of a compound at levels lower than those required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved. If desired, the effective dailydose can be divided into multiple doses for purposes of administration.Consequently, single dose compositions can contain such amounts orsubmultiples thereof to make up the daily dose. The dosage can beadjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products. In further various aspects, a preparation canbe administered in a “prophylactically effective amount”; that is, anamount effective for prevention of a disease or condition.

As used herein, the terms “age-related disorder” or “age-relateddisease” refers to disorders or diseases in which aging is a major riskfactor. In some aspects, age-related diseases or disorders can be basedon disease type, and can include three main types: (1) abnormalproliferative diseases, such as cancer; (2) degenerative diseases,including neuron degenerating disease (Alzheimer's, Parkinson's,stroke), myocardial infarction, heart failure, atherosclerosis,hypertension, osteoarthritis, osteoporosis, sarcopenia, loss of bonemarrow, Rheumatoid arthritis, degraded immune function, diabetes,Idiopathic pulmonary fibrosis, age-related macular degeneration; and (3)function decreasing disorders, including declines in testosterone,estrogen, growth hormone, IGF-I, reduced energy production and so on. Inother aspects, age-related diseases or disorders can also be classifiedbased on the type of cells involved, and can include two main classes:(1) in postmitotic cells: neuron degeneration (Alzheimer's, Parkinson's,stroke), sarcopenia (loss of muscle), cardiovascular diseases (heartfailure, myocardial infarction); and (2) in mitotic cells: loss of bonemarrow, degraded immune function, diabetes, idiopathic pulmonaryfibrosis, age-related macular degeneration, rheumatoid arthritis,osteoarthritis, osteoporosis, atherosclerosis, and hypertension. Infurther aspects, age-related diseases or disorders associated withmitochondrial dysfunction or/and telomere dysfunction include, but arenot limited to, cancer, osteoarthritis, age-related maculardegeneration, idiopathic pulmonary fibrosis (IPF), Parkinson's disease,Alzheimer's disease, Huntington's disease, skin aging, cataract,multiple sclerosis, Sjogren, Rheumatoid arthritis, atherosclerosis,myocardial infarction, heart failure, hypertension, stroke, diabetesmellitus, osteoporosis, obesity, grey hair, hearing loss, and the like.In still further aspects, the present invention encompasses, but is notlimited to the foregoing diseases or disorders.

As used herein, “telomere disorder” refer to a disease or disordercharacterized by or exhibiting a telomere dysfunction. In furthervarious aspects, the dysfunction is telomere shortening. In furthervarious aspects, a telomere disorder can comprise an immune-mediateddisorder, inflammatory disorder, or chronic pulmonary disorder, such as,for example, idiopathic pulmonary fibrosis.

As used herein, the term “senescence” refers to a cell cycle-arrestedstate in mitotic cells, which can be induced by telomere dysfunction,DNA damage, or oncogene activation. In budding yeast, senescent cellscaused by telomere dysfunction are arrested at the G2/M phase of thecell cycle. In mammalian cells, senescent cells are arrested at the G0phase, which is a non-dividing phase outside of the cell cycle. Forexample, senescence in fibroblasts means that cells show no increase innumber under the microscope for at least 4 days after passage andexhibit β-galactosidase positive staining.

As used herein, the term “post-mitotic cells” refers to a group of cellsthat are in arrested state at G0, which is a non-dividing phase outsideof the cell cycle, but continue to perform their main functions for therest of the organism's life. Post-mitotic cells include neuronal cells,heart muscle cells, and muscle cells. Some cell types in matureorganisms, such as parenchymal cells of the liver and kidney, enter theG0 phase semi-permanently and can only be induced to begin dividingagain under very specific circumstances. In various aspects, these typesof cells can also be considered as post-mitotic cells when they are inG0 phase.

As used herein, “IC₅₀,” is intended to refer to the concentration of asubstance (e.g., a compound or a drug) that is required for 50%inhibition of a biological process, or component of a process, includinga protein, subunit, organelle, ribonucleoprotein, etc. In one aspect, anIC₅₀ can refer to the concentration of a substance that is required for50% inhibition in vivo, as further defined elsewhere herein. In afurther aspect, IC₅₀ refers to the half maximal (50%) inhibitoryconcentration (IC) of a substance.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner.

As used herein, the term “derivative” refers to a compound having astructure derived from the structure of a parent compound (e.g., acompound disclosed herein) and whose structure is sufficiently similarto those disclosed herein and based upon that similarity, would beexpected by one skilled in the art to exhibit the same or similaractivities and utilities as the claimed compounds, or to induce, as aprecursor, the same or similar activities and utilities as the claimedcompounds. Exemplary derivatives include salts, esters, amides, salts ofesters or amides, and N-oxides of a parent compound.

Compounds described herein may comprise atoms in both their naturalisotopic abundance and in non-natural abundance. The disclosed compoundscan be isotopically-labeled or isotopically-substituted compoundsidentical to those described, but for the fact that one or more atomsare replaced by an atom having an atomic mass or mass number differentfrom the atomic mass or mass number typically found in nature. Examplesof isotopes that can be incorporated into compounds of the inventioninclude isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous,fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N ¹⁸O, ¹⁷O, ³⁵S, ¹⁸Fand ³⁶Cl, respectively. Compounds further comprise prodrugs thereof, andpharmaceutically acceptable salts of said compounds or of said prodrugswhich contain the aforementioned isotopes and/or other isotopes of otheratoms are within the scope of this invention. Certainisotopically-labeled compounds of the present invention, for examplethose into which radioactive isotopes such as ³H and ¹⁴ C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances.Isotopically labeled compounds of the present invention and prodrugsthereof can generally be prepared by carrying out the procedures below,by substituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent.

The compounds described in the invention can be present as a solvate. Insome cases, the solvent used to prepare the solvate is an aqueoussolution, and the solvate is then often referred to as a hydrate. Thecompounds can be present as a hydrate, which can be obtained, forexample, by crystallization from a solvent or from aqueous solution. Inthis connection, one, two, three or any arbitrary number of solvate orwater molecules can combine with the compounds according to theinvention to form solvates and hydrates. Unless stated to the contrary,the invention includes all such possible solvates.

The term “co-crystal” means a physical association of two or moremolecules which owe their stability through non-covalent interaction.One or more components of this molecular complex provide a stableframework in the crystalline lattice. In certain instances, the guestmolecules are incorporated in the crystalline lattice as anhydrates orsolvates, see e.g., Almarasson, O., et. al. (2004) The Royal Society ofChemistry, 1889-1896. Examples of co-crystals include p-toluenesulfonicacid and benzenesulfonic acid.

It is known that chemical substances form solids which are present indifferent states of order which are termed polymorphic forms ormodifications. The different modifications of a polymorphic substancecan differ greatly in their physical properties. The compounds accordingto the invention can be present in different polymorphic forms, with itbeing possible for particular modifications to be metastable. Unlessstated to the contrary, the invention includes all such possiblepolymorphic forms.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989).

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

B. Compounds

In one aspect, the invention relates to compounds and compositions foruse as anti-aging agents for the treatment of age-related disorders ordiseases. In a further aspect, the invention relates to compounds andcompositions for use in the treatment of age-related diseases andage-related disorders. More specifically, in one aspect, the presentinvention relates to compounds and compositions for the treatment oftumorigenesis, and malignant cancer development, myocardial infarction,cerebellar infarction, stroke, Parkinson's disease, heart failure,atherosclerosis, hypertension, cataract, age-related maculardegeneration, sarcopenia, osteoarthritis, osteoporosis, loss of bonemarrow, multiple sclerosis, Sjogren, Rheumatoid arthritis, decreasedimmune function, diabetes, idiopathic pulmonary fibrosis, andneurodegenerating disease, Alzheimer's disease, Huntington's disease,and disorders caused by dysfunction in testosterone, estrogen, growthhormone, IGF-I, or energy production.

It is contemplated that each disclosed derivative can be optionallyfurther substituted. It is also contemplated that any one or morederivative can be optionally omitted from the invention. It isunderstood that the disclosed compounds can be employed in the disclosedmethods of using.

In one aspect, the invention relates to lithium compounds or apharmaceutically acceptable salt thereof. In a further aspect, thelithium compound, or a pharmaceutically acceptable salt thereof isselected from lithium chloride, lithium bromide, lithium carbonate,lithium nitrate, lithium sulfate, lithium acetate, lithium lactate,lithium citrate, lithium aspartate, lithium gluconate, lithium malate,lithium ascorbate, lithium orotate, lithium succinate, and combinationsthereof. In a still further aspect, the lithium compound, or apharmaceutically acceptable salt thereof is selected from lithiumchloride, lithium bromide, lithium carbonate, lithium citrate, andlithium orotate.

In one further aspect, the lithium compound, or a pharmaceuticallyacceptable salt thereof is lithium chloride. In a further aspect, thelithium compound, or a pharmaceutically acceptable salt thereof islithium bromide. In a still further aspect, the lithium compound, or apharmaceutically acceptable salt thereof is lithium carbonate. In a yetfurther aspect, the lithium compound, or a pharmaceutically acceptablesalt thereof is lithium citrate. In an even further aspect, the lithiumcompound, or a pharmaceutically acceptable salt thereof is lithiumorotate.

In a further aspect, the invention relates to glycyrrhizie triterpenoidcompounds or a pharmaceutically acceptable salt thereof. In a stillfurther aspect, glycyrrhizie triterpenoid compounds include compounds,such as, for example, carbenoxolone, cicloxolone, glycyrrhizin and itsaglycone derivative, enoxolone, as well as, analogues and salts thereof.In a yet further aspect, glycyrrhizin refers to(3β,20β)-20-carboxy-11-oxo-30-norolean-12-en-3-yl2-O-β-D-glucopyranuronosyl-α-D-glucopyranosiduronic acid, and can beused interchangeably with glycyrrhizic acid and glycyrrhizinic acid. Itis a compound having the structure represented by the formula:

In a further aspect, enoxolone refers(2S,4aS,6aS,6bR,8aR,10S,12aS,12bR,14bR)-10-hydroxy2,4a,6a,6b,9,9,12a-heptamethyl-13-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-2-carboxylicacid, and can be used interchangeably with glycyrrhetinic acid orglycyrrhetic acid. It is commonly obtained from the hydrolysis ofglycyrrhizic acid. In a still further aspect, enoxolone is a compoundhaving the structure represented by the formula:

In a further aspect, glycyrrhizic acid,20-B-carboxy-11-oxo-30-norolean-12-en-3B-yl-2-O-B-D-glucopyranuronsyl-a-D-glucopyranosiduronicacid, which can also be known as glycyrrhizin, glycyrrhizinic acid orglycyrrhetinic acid glycoside (also referred to as biosone, enoxolone,and glycyrrhetin), an extract from glycyrrhiza, better known aslicorice, an extract of the dried rhizome and roots of Glycyrrhizaglabra, is an exemplary triterpene according to the present invention.In a yet further aspect, analogous triterpenes can include carbenoxoloneand cicloxolone. In a still further aspect, the invention relates toglycyrrhizic acid and analogues thereof, in the form of acids, salts,esters and other derivatives. In an even further aspect, derivatives caninclude, such as, for example: Glycyrrhisoflavone; Benzylglycyrrhetinate; Glycyrrhizinic acid ammonium salt; Glycyrrhizinic acidhydrolase; Glycyrrhizinic acid; Zinc glycyrrhizinate; Glycyrrhetic acid3-0-(hydrogen sulfate); Glycyrrhetinyl stearate; Monopotassiumglycyrrhetin; 3-Oxo-18-a-glycyrrhetinic acid; 11-Deoxoglycyrrhetinicacid hydrogen maleate sodium salt; 11-Deoxoglycyrrhetinic acid hydrogenmaleate; 18-a-Glycyrrhizic acid diammonium salt; Monoarginineglycyrrhizinate; Potassium glycyrrhizinate; 18-a-Glycyrrhizinic acid;18-a-Glycyrrhizic acid monosodium salt; 18-a-Glycyrrhizic aciddipotassium salt; 18-a-Glycyrrhizic acid disodium salt;18-a-Glycyrrhizic acid monopotassium salt; Glycyrrhizinic acidmono-triethanolamine) salt; Glycyrrhizinic acid disodium salt; Trisodiumglycyrrhizinate; Dipotassium glycyrrhizate; Triammonium glycyrrhizinate;Dioxoglycyrrhetinic acid; Sodium glycyrrhizate; Ammoniumglycyrrhizinate; Tripotassium glycyrrhizate; Glycyrrhetinic acidnicotinate morpholine salt; Benzyl 3-0-benzyl-18-Bglycyrrhetate;3-0-Acetylglycyrrhetaldehyde; Glycyrrhizic acid monopotassium salt;Magnesium di-3-acetyl-18-B-glycyrrhetinate; Magnesium monomethoxymono-3-acetyl-18-B-glycyrrhetinate; Dehydrocorydaline glycyrrhizate;Ruscogenin acetylglycyrrhetinate; Glycyrrhetic acid 3-0-glucuronide;Cinoxolone; crotyl glycyrrhetate; Glycyrrhizin trimethyl ester;3-0-Acetylglycyrrhetoyl chloride; Aluminum acetylglycyrrhetate;Dipotassium glycyrrhetinate; Methyl 11-deoxo-18-aglycyrrhetate; Calciumpotassium glycyrrhizinate; Glycyrrhetic acid monophosphate;B-glycyrrhetinicacid 3-acetate; 24-Hydroxyglycyrrhetic acid;3-0-Acetyl-18-B-glycyrrhetamide; Deoxyglycyrrhetic acid;N-(18-B-Glycyrrhetyl)glycine; 3 -0-Propionyl-18-B-glycyrrhetic acid;o-(18-B-Glycyrrhetamideo)benzoic acid; Glycyrrhetol; Stearylglycyrrhetinate; Glycyrrhizic acid monosodium salt; Methyl3-0-acetylglycyrrhetinate; Carbenoxolone sodium; Ammoniumglycyrrhetinate; 3 -Dehydro-18-B-glycyrrhetic acid; 3-0-Acetyl-18-B-glycyrrhetic acid; Methyl 3-0-toxylglycyrrhetate;Biogastrone; Glycyrrhetinic acid phthalate; Lauroyl glycyrrhetinate;Methyl 3-oxoglycyrrhetate; Methyl-B-glycyrrhetinate; Sodiumglycyrrhetinate; Methyl glycyrrhetate; 18-a-Glycyrrhetic acid;Glycyrrhizin; 11-Deoxo-18-B-glycyrrhetic acid; Glycyrrhetic acid; andCarbenoxolone sodium.

In a further aspect, the least one glycyrrhizie triterpenoid compound isselected from glycyrrhizin, enoxolone, carbenoxolone, cicloxolone,pharmaceutically acceptable salts thereof, and combinations thereof. Ina still further aspect, the least one glycyrrhizie triterpenoid isglycyrrhizin, having the structure represented by the formula:

In a further aspect, the invention relates to retinoic acid compounds ora pharmaceutically acceptable salt thereof. In a still further aspect,the retinoic acid compound can comprise all-trans-retinoic acid or acis-retinoic acid, or pharmaceutically acceptable salts thereof, andcombinations thereof. In yet further aspects, the retinoic compoundsinclude compounds, such as, for example, 7-cis-retinoic acid,9-cis-retinoic acid, 11-cis-retinoic acid, 13-cis-retinoic acid, orpharmaceutically acceptable salts thereof, and combinations thereof.

In a further aspect, the lithium compound, or a pharmaceuticallyacceptable salt thereof is selected from lithium chloride, lithiumbromide, lithium carbonate, lithium nitrate, lithium sulfate, lithiumacetate, lithium lactate, lithium citrate, lithium aspartate, lithiumgluconate, lithium malate, lithium ascorbate, lithium orotate, lithiumsuccinate, and combinations thereof; and the glycyrrhizie triterpenoidcompound is selected from glycyrrhizin, enoxolone, carbenoxolone,cicloxolone, pharmaceutically acceptable salts thereof, and combinationsthereof.

In a still further aspect, the lithium compound, or a pharmaceuticallyacceptable salt thereof is selected from lithium chloride, lithiumbromide, lithium carbonate, lithium citrate, and lithium orotate; andthe least one glycyrrhizie triterpenoid compound is selected fromglycyrrhizin, enoxolone, carbenoxolone, cicloxolone, pharmaceuticallyacceptable salts thereof, and combinations thereof.

In a yet further aspect, the lithium compound, or a pharmaceuticallyacceptable salt thereof is selected from lithium chloride, lithiumbromide, lithium carbonate, lithium nitrate, lithium sulfate, lithiumacetate, lithium lactate, lithium citrate, lithium aspartate, lithiumgluconate, lithium malate, lithium ascorbate, lithium orotate, lithiumsuccinate, and combinations thereof; and the glycyrrhizie triterpenoidcompound is selected from glycyrrhizin, and pharmaceutically acceptablesalts thereof.

In an even further aspect, the one lithium compound, or apharmaceutically acceptable salt thereof is selected from lithiumchloride, lithium bromide, lithium carbonate, lithium citrate, andlithium orotate; and the glycyrrhizie triterpenoid compound is selectedfrom glycyrrhizin, and pharmaceutically acceptable salts thereof.

In a further aspect, the lithium compound, or a pharmaceuticallyacceptable salt thereof is selected from lithium chloride, lithiumbromide, lithium carbonate, lithium citrate, and lithium orotate; andthe glycyrrhizie triterpenoid compound is selected from glycyrrhizin,and pharmaceutically acceptable salts thereof.

In a further aspect, the lithium compound, or a pharmaceuticallyacceptable salt thereof is selected from lithium chloride, lithiumbromide, lithium carbonate, lithium citrate, and lithium orotate; theglycyrrhizie triterpenoid compound is selected from glycyrrhizin, andpharmaceutically acceptable salts thereof; and the retinoic acidcompound or a pharmaceutically acceptable salt thereof is selected fromall-trans-retinoic 7-cis-retinoic acid, 9-cis-retinoic acid,11-cis-retinoic acid, 13-cis-retinoic acid, and pharmaceuticallyacceptable salts thereof.

C. Methods for Age-Related Diseases and Disorders

In one aspect, the invention relates to methods for preventing cellularaging in a cell of a subject, the method comprising the step ofproviding to the cell an effective amount of at least one lithiumcompound or a pharmaceutically acceptable salt thereof, and an effectiveamount of at least one glycyrrhizie triterpenoid compound or apharmaceutically acceptable salt thereof, thereby preventing the cellfrom aging. In further aspects, the method can further comprise the stepof providing to the cell an effective amount of at least one retinoicacid compound or a pharmaceutically acceptable salt thereof.

In some aspects, the cellular anti-aging activity can comprisemaintaining cell replication potential; maintaining senescence,maintaining cell cycle-arrested state in post-mitotic cells,stimulating, improving, or maintaining mitochondrial function;preventing deterioration of mitochondria, preventing cell deathfollowing senescence deterioration, or a combination thereof. In otheraspects, the cellular anti-aging activity can comprise cell rejuvenationor survival.

In other aspects, the cellular anti-aging activity can comprise activityagainst at least one component of the TOR (Target of rapamycin) pathway,IGF-I (insulin-like growth factors) receptor pathway, EGFR (epidermalgrowth factor receptor) pathway, or a combination thereof. In a furtheraspect, the cellular anti-aging activity can comprise inhibition of atleast one component of the TOR (Target of rapamycin) pathway, IGF-I(insulin-like growth factors) receptor pathway, EGFR (epidermal growthfactor receptor) pathway, or a combination thereof. In a still furtheraspect, the at least one component can comprise an enzyme or protein.

In a further aspect, the cellular anti-aging activity comprisespreventing an age-related disease or disorder. In a still furtheraspect, the age-related disease or disorder is associated withage-related cell loss, loss of mitochondrial function, or loss oftelomere function. In a still further aspect, the age-related disease ordisorder can comprise an abnormal proliferative disease, a degenerativedisease, or a function-decreasing disorder. In a yet further aspect, theage-related disease or disorder can comprise tumorigenesis, andmalignant cancer development, myocardial infarction, cerebellarinfarction, stroke, Parkinson's disease, heart failure, atherosclerosis,hypertension, cataract, age-related macular degeneration, sarcopenia,osteoarthritis, osteoporosis, loss of bone marrow, multiple sclerosis,Sjogren, Rheumatoid arthritis, decreased immune function, diabetes,idiopathic pulmonary fibrosis, a neurodegenerating disease, Alzheimer'sdisease, Huntington's disease, and disorders caused by dysfunction intestosterone, estrogen, growth hormone, IGF-I, or energy production.

To treat or control the cellular aging activity, the compounds andpharmaceutical compositions comprising the compounds are provided to acell, such as a mammalian cell, e.g., a human cell. In a further aspect,providing can comprise administering or contacting the cell with thecompounds or compositions. In a still further aspect, prior to providingthe compounds or compositions, the cell can be identified with a needfor treatment of anti-aging treatment, as described herein.

In one aspect, the lithium compound and the glycyrrhizie triterpenoidcompound are used at low doses. In a further aspect, the lithiumcompound and the glycyrrhizie triterpenoid compound dose is from about0.001 to about 10000 μM in serum medium. In a still further aspect, theeffective amount of the lithium compound is from about 0.01 to about 100μM in serum medium, including exemplary subranges of about 0.1 to about10, and about 0.2 to about 1 μM. In an even further aspect, theeffective amount of the glycyrrhizie triterpenoid compound is from about0.1 to about 1000 μM in serum medium, including exemplary subranges ofabout 1 to about 100, and about 1 to about 50 μM.

In a further aspect, the effective amount of the lithium compound isfrom about 0.1 to about 10 μM in serum medium and the effective amountof the glycyrrhizie triterpenoid compound is from about 1 to about 1000μM in serum medium. In an even further aspect, the effective amount ofthe lithium compound is from about 0.1 to about 1 μM in serum medium andthe effective amount of the glycyrrhizie triterpenoid compound is fromabout 1 to about 100 μM in serum medium. In a still further aspect, theeffective amount of the lithium compound is from about 0.1 to about 0.5μM in serum medium and the effective amount of the glycyrrhizietriterpenoid compound is from about 1 to about 50 μM in serum medium. Ina yet further aspect, the ratio of lithium compound to glycyrrhizietriterpenoid compound is from about 1:10 to about 1:1000.

In one aspect, the retinoic acid compound is also used at low doses. Ina further aspect, the retinoic acid compound dose can be from about0.001 to about 10000 μM in serum medium. In a still further aspect, theeffective amount of the retinoic acid compound is from about 0.01 toabout 100 μM in serum medium, including exemplary subranges of about 0.1to about 10, and about 0.2 to about 1 μM. In an even further aspect, theeffective amount of the retinoic acid compound is from about 0.1 toabout 1000 μM in serum medium, including exemplary subranges of about 1to about 100, and about 1 to about 50 μM.

In a further aspect, the effective amount of the retinoic acid compoundis from about 0.1 to about 10 μM in serum medium and the effectiveamount of the glycyrrhizie triterpenoid compound is from about 1 toabout 1000 μM in serum medium. In an even further aspect, the effectiveamount of the retinoic acid compound is from about 0.1 to about 1 μM inserum medium and the effective amount of the retinoic acid compound isfrom about 1 to about 100 μM in serum medium. In a still further aspect,the effective amount of the retinoic acid compound is from about 0.1 toabout 0.5 μM in serum medium and the effective amount of the retinoicacid compound is from about 1 to about 50 μM in serum medium.

In various aspects, it has been surprising discovered that thecombination of the lithium compound and the glycyrrhizie triterpenoidcompound at these low doses and ratios exhibit the disclosed novelanti-aging activity. In a further aspect, the anti-aging activity is notexhibited when the lithium compound and the glycyrrhizie triterpenoidcompound are used as monotherapy. In a still further aspect, thesynergistic anti-aging activity of the lithium compound and theglycyrrhizie triterpenoid compound is not present at higher doses.

In another aspect, the invention also relates to methods for preventingcellular aging activity in a subject, the method comprising the step ofproviding to the subject an effective amount of at least oneintracellular alkalinizing agent or a pharmaceutically acceptable saltthereof, thereby preventing the cellular aging activity.

In a further aspect, the intracellular alkalization action comprisesdirect competition with Na+ on a sodium-hydrogen exchanger, increasedexpression or number of sodium-hydrogen exchangers, intracellular Na+retention; increasing membrane permeability for Na+, increased Na+reabsorption; increased secretion of H+, decreased accumulation of acidproducts of metabolism; or a combination thereof. In a still furtheraspect, the method can further comprise providing to the cell aneffective amount of at least one agent for improving telomere functionor a pharmaceutically acceptable salt thereof.

In a yet further aspect, the step of providing to the subject comprisesproviding to the subject an effective amount of at least one lithiumcompound or a pharmaceutically acceptable salt thereof, and an effectiveamount of at least one glycyrrhizie triterpenoid compound or apharmaceutically acceptable salt thereof. In various aspects, andwithout wishing to be bound by a particular theory, it is believed thatthe lithium and glycyrrhizie triterpenoid compound increaseintracellular pH, which thus exhibit anti-aging activity.

To treat or control the age-related disease or disorder, the compoundsand pharmaceutical compositions comprising the compounds areadministered to a subject in need thereof, such as a vertebrate, e.g., amammal, a fish, a bird, a reptile, or an amphibian. The subject can be ahuman, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow,cat, guinea pig or rodent. The term does not denote a particular age orsex. Thus, adult and newborn subjects, as well as fetuses, whether maleor female, are intended to be covered. The subject is preferably amammal, such as a human.

In a further aspect, the subject has been diagnosed with a need foranti-aging treatment prior to the administering step. In a still furtheraspect, the subject has been diagnosed with an age-related disease ordisorder associated with age-related cell loss, loss of mitochondrialfunction, or loss of telomere function. Prior to administering thecompounds or compositions, the subject can be diagnosed with a need fortreatment of age-related disorder or disease, such as idiopathicpulmonary fibrosis (IPF). In a still further aspect, the subject can beidentified with a need for treatment of anti-aging treatment, asdescribed herein.

The compounds or compositions can be administered to the subjectaccording to any method. Such methods are well known to those skilled inthe art and include, but are not limited to, oral administration,transdermal administration, administration by inhalation, nasaladministration, topical administration, intravaginal administration,ophthalmic administration, intraaural administration, intracerebraladministration, rectal administration, sublingual administration, buccaladministration and parenteral administration, including injectable suchas intravenous administration, intra-arterial administration,intramuscular administration, and subcutaneous administration.Administration can be continuous or intermittent. A preparation can beadministered therapeutically; that is, administered to treat an existingdisease or condition. A preparation can also be administeredprophylactically; that is, administered for prevention of a disease orcondition.

The effective amount or dosage of the compounds can vary within widelimits. Such a dosage is adjusted to the individual requirements in eachparticular case including the specific compound(s) being administered,the route of administration, the condition being treated, as well as thepatient being treated. In general, in the case of oral or parenteraladministration to adult humans weighing approximately 70 Kg or more, adaily dosage of about 1 mg to about 10,000 mg, preferably from about 2mg to about 1,000 mg, should be appropriate, although the upper limitmay be exceeded. The daily dosage can be administered as a single doseor in divided doses, or for parenteral administration, as a continuousinfusion. Single dose compositions can contain such amounts orsubmultiples thereof of the compound or composition to make up the dailydose. The dosage can be adjusted by the individual physician in theevent of any contraindications. Dosage can vary, and can be administeredin one or more dose administrations daily, for one or several days. Insome aspects, the compounds can be administered on a regimen of 1 to 4times per day, preferably once or twice per day. This dosing regimen canbe adjusted to provide the optimal therapeutic response.

In one aspect, the lithium compound and the glycyrrhizie triterpenoidcompound are used at low doses in a subject. In a further aspect, thelithium compound and the glycyrrhizie triterpenoid compound dose is fromabout 0.1 mg to about 10,000 mg in a subject. In a still further aspect,the effective amount of the lithium compound is from about 0.1 mg toabout 100 mg in a subject, including exemplary subranges of about 1 toabout 50 mg, and about 2 to about 25 mg. In an even further aspect, theeffective amount of the glycyrrhizie triterpenoid compound is from about1 to about 1,000 mg in a subject, including exemplary subranges of about1 to about 100 mg, and about 1 to about 50 mg.

In a further aspect, the effective amount of the lithium compound isfrom about 0.1 mg to about 100 mg and the effective amount of theglycyrrhizie triterpenoid compound is from about 1 to about 1000 mg in asubject. In an even further aspect, the effective amount of the lithiumcompound is from about 1 mg to about 50 mg and the effective amount ofthe glycyrrhizie triterpenoid compound is from about 1 to about 100 mg.In a still further aspect, the effective amount of the lithium compoundis from about 1 to about 25 mg and the effective amount of theglycyrrhizie triterpenoid compound is from about 1 to about 50 mg. In ayet further aspect, the ratio of lithium compound to glycyrrhizietriterpenoid compound is from about 1:10 to about 1:1000.

In a further aspect, the retinoic acid compound is also used at lowdoses in a subject. In a further aspect, the retinoic acid compound doseis from about 0.1 mg to about 10,000 mg in a subject. In a still furtheraspect, the effective amount of the retinoic acid compound is from about0.1 mg to about 100 mg in a subject, including exemplary subranges ofabout 1 to about 50 mg, and about 2 to about 25 mg. In an even furtheraspect, the effective amount of the retinoic acid compound is from about1 to about 1,000 mg in a subject, including exemplary subranges of about1 to about 100 mg, and about 1 to about 50 mg.

In determining the effective dose or dosage of the pharmaceuticalcomposition of the invention, a response to a prophylactic and/ortreatment method of the invention can, for example, also be measured bydetermining the physiological effects of the treatment or medication,such as the decrease or lack of disease symptoms followingadministration of the treatment or pharmacological agent. Other assayswill be known to one of ordinary skill in the art and can be employedfor measuring the level of the response. For example, the diagnosticmethods that are used to ascertain the likelihood that a subject has anage-related disorder or disease can be used to ascertain the level ofresponse to a prophylactic and/or treatment method of the invention. Theamount of a treatment may be varied for example by increasing ordecreasing the amount of a therapeutic composition, by changing thetherapeutic composition administered, by changing the route ofadministration, by changing the dosage timing and so on. The effectiveamount will vary with the particular condition being treated, the ageand physical condition of the subject being treated, the severity of thecondition, the duration of the treatment, the nature of the concurrenttherapy (if any), the specific route of administration, and the likefactors within the knowledge and expertise of the health practitioner.For example, an effective amount can depend upon the degree to which anindividual has abnormal levels and/or activity of a pathway associatedprotein or pathway associated protein complex.

The factors involved in determining an effective amount are well knownto those of ordinary skill in the art and can be addressed with no morethan routine experimentation. It is generally preferred that a maximumdose of the pharmacological agents of the invention (alone or incombination with other therapeutic agents) be used, that is, the highestsafe dose according to sound medical judgment. It will be understood bythose of ordinary skill in the art however, that a patient may insistupon a lower dose or tolerable dose for medical reasons, psychologicalreasons or for virtually any other reasons.

In some aspects, the effective amount is a therapeutically effectiveamount. In other aspects, the effective amount is a prophylacticallyeffective amount. In further aspects, the subject is a mammal. In stillfurther aspects, the mammal is a human.

In a further aspect, the method further comprises the step ofidentifying a subject in need of anti-aging treatment. In a stillfurther aspect, the subject in need of anti-aging treatment compriseshaving at least one risk factor for developing an age-related disease ordisorder. In a yet further aspect, the age-related disease or disorderis associated with age-related cell loss, loss of mitochondrialfunction, or loss of telomere function. In an even further aspect, theage-related disease or disorder comprises an abnormal proliferativedisease, a degenerative disease, or a function-decreasing disorder. Inan even further aspect, the age-related disease or disorder is selectedfrom tumorigenesis, malignant cancer development, myocardial infarction,cerebellar infarction, stroke, Parkinson's disease, heart failure,atherosclerosis, hypertension, cataract, age-related maculardegeneration, sarcopenia, osteoarthritis, osteoporosis, loss of bonemarrow, multiple sclerosis, Sjogren, Rheumatoid arthritis, decreasedimmune function, diabetes, idiopathic pulmonary fibrosis, aneurodegenerating disease, Alzheimer's disease, Huntington's disease,and disorders caused by dysfunction in testosterone, estrogen, growthhormone, IGF-I, or energy production.

In another aspect, the invention also relates to a method for thetreatment of a subject, the method comprising the steps of: diagnosingthe subject as having an age-related disorder or disease; andadministering to the subject an effective amount of at least one lithiumcompound or a pharmaceutically acceptable salt thereof, and an effectiveamount of at least one glycyrrhizie triterpenoid compound or apharmaceutically acceptable salt thereof.

In another aspect, a method of diagnosis comprises performing anexperiment upon the subject and identifying a level of a biologicalmarker. In a further aspect diagnosing comprises determining, in apatient, levels of a marker (e.g., gene expression) indicative of astate of the patient, the state being predictive as to whether thepatient will manifest reduced symptoms in response to a treatment.

In a further aspect, the biological marker is a marker for anage-related disease or disorder. In a still further aspect, the subjectis a biological sample. In a still further aspect, the biological sampleis selected from a cell, blood, saliva, urine, tissue, or phlegm.

In one aspect, diagnosis of an age-related disorder or disease comprisesa medical history. In a further aspect, the diagnosis comprisescomparing the findings of the medical history with the diagnosticstandards. In a still further aspect, the diagnosis comprises finding ofat least one risk factor for developing an age-related disease ordisorder. In a yet further aspect, the age-related disease or disorderis associated with age-related cell loss, loss of mitochondrialfunction, or loss of telomere function. In an even further aspect, theage-related disease or disorder comprises an abnormal proliferativedisease, a degenerative disease, or a function-decreasing disorder.

D. Anti-Aging Compositions

In one aspect, the invention relates to compositions comprising aneffective amount of at least one lithium compound or a pharmaceuticallyacceptable salt thereof and an effective amount of at least oneglycyrrhizie triterpenoid compound or a pharmaceutically acceptable saltthereof. In further aspects, the compositions can comprise an effectiveamount of at least one retinoic acid compound or a pharmaceuticallyacceptable salt thereof. In some aspects, the composition can comprisepharmaceutical compositions. In other aspects, the composition cancomprise nutraceutical compositions.

The compounds have anti-aging activity, and generally have IC₅₀ valuesranging from about 0.01 micromolar to about 100 millimolar. IC₅₀ refersto the concentration of the compound that is required for 50% antagonismor inhibition of the target in vitro. IC₅₀ also refers to theconcentration of a substance that is required for 50% antagonism orinhibition of the target in vivo. The activity of the compounds,including IC₅₀, is determined according to the procedures discussedbelow in the Examples section.

Pharmaceutically acceptable salts of the compounds are conventionalacid-addition salts or base-addition salts that retain the biologicaleffectiveness and properties of the compounds and are formed fromsuitable non-toxic organic or inorganic acids or organic or inorganicbases. Exemplary acid-addition salts include those derived frominorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, andthose derived from organic acids such as p-toluenesulfonic acid,salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citricacid, malic acid, lactic acid, fumaric acid, and the like. Examplebase-addition salts include those derived from ammonium, potassium,sodium and, quaternary ammonium hydroxides, such as for example,tetramethylammonium hydroxide. Chemical modification of a pharmaceuticalcompound into a salt is a known technique to obtain improved physicaland chemical stability, hygroscopicity, flowability and solubility ofcompounds. See, e.g., Ansel, H. et. al., Pharmaceutical Dosage Forms andDrug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.

The pharmaceutical and nutraceutical compositions comprise the compoundsin a pharmaceutically acceptable carrier. A pharmaceutically acceptablecarrier refers to sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, as well as sterile powders for reconstitutioninto sterile injectable solutions or dispersions just prior to use.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol and the like), carboxymethylcellulose andsuitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. The compounds can beformulated with pharmaceutically acceptable carriers or diluents as wellas any other known adjuvants and excipients in accordance withconventional techniques such as those disclosed in Remington: TheScience and Practice of Pharmacy, 19th Edition, Gennaro, Ed., MackPublishing Co., Easton, Pa., 1995.

In various aspects, the disclosed pharmaceutical and nutraceuticalcompositions comprise the disclosed compounds (includingpharmaceutically acceptable salt(s) thereof) as an active ingredient, apharmaceutically acceptable carrier, and, optionally, other therapeuticingredients or adjuvants. The instant compositions include thosesuitable for oral, rectal, topical, and parenteral (includingsubcutaneous, intramuscular, and intravenous) administration, althoughthe most suitable route in any given case will depend on the particularhost, and nature and severity of the conditions for which the activeingredient is being administered. The compositions can be convenientlypresented in unit dosage form and prepared by any of the methods wellknown in the art of pharmacy.

Pharmaceutical compositions of the present invention suitable forparenteral administration can be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical and nutraceutical compositions of the present inventioncan be in a form suitable for topical use such as, for example, anaerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles,and the like. Further, the compositions can be in a form suitable foruse in transdermal devices. These formulations can be prepared,utilizing a compound of the invention, or pharmaceutically acceptablesalts thereof, via conventional processing methods. As an example, acream or ointment is prepared by mixing hydrophilic material and water,together with about 5 wt % to about 10 wt % of the compound, to producea cream or ointment having a desired consistency.

Pharmaceutical and nutraceutical compositions of this invention can bein a form suitable for rectal administration wherein the carrier is asolid. It is preferable that the mixture forms unit dose suppositories.Suitable carriers include cocoa butter and other materials commonly usedin the art. The suppositories can be conveniently formed by firstadmixing the composition with the softened or melted carrier(s) followedby chilling and shaping in molds.

In various aspects, the pharmaceutical and nutraceutical compositions ofthis invention can include a pharmaceutically acceptable carrier and acompound or a pharmaceutically acceptable salt of the compounds of theinvention. The compounds of the invention, or pharmaceuticallyacceptable salts thereof, can also be included in pharmaceutical andnutraceutical compositions in combination with one or more othertherapeutically active compounds.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media can be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likecan be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like can be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets can be coated by standard aqueous or nonaqueoustechniques.

A tablet containing the composition of this invention can be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets can be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above can include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining a compound of the invention, and/or pharmaceuticallyacceptable salts thereof, can also be prepared in powder or liquidconcentrate form.

In a further aspect, the effective amount is a therapeutically effectiveamount. In a still further aspect, the effective amount is aprophylactically effective amount.

In a further aspect, the composition is formulated for parenteraladministration. In a still further aspect, the composition is formulatedfor inhalation. In yet a further aspect, the composition is formulatedfor oral administration. In an even further aspect, the composition isformulated for topical administration.

It is understood that the disclosed compositions can be prepared fromthe disclosed compounds. It is also understood that the disclosedcompositions can be employed in the disclosed methods of using.

E. Kits

In one aspect, the invention relates to a kit comprising an effectiveamount of at least one lithium compound or a pharmaceutically acceptablesalt thereof, an effective amount of at least one glycyrrhizietriterpenoid compound or a pharmaceutically acceptable salt thereof, andone or more of: a) at least one agent known to treat an age-relateddisorder or disease; b) instructions for treating the age-relateddisorder or disease; and c) instructions for administering the lithiumcompound and the glycyrrhizie triterpenoid compound in connection withthe age-related disorder or disease. In a further aspect, the kit cancomprise an effective amount of at least one retinoic acid compound or apharmaceutically acceptable salt thereof, and instructions foradministering the retinoic acid compound in connection with theage-related disorder or disease.

The kits can also comprise compounds co-packaged, co-formulated, and/orco-delivered with other components. For example, a drug manufacturer, adrug reseller, a physician, a compounding shop, or a pharmacist canprovide a kit comprising a disclosed compound and another component fordelivery to a patient.

In a further aspect, the age-related disorder or disease is associatedwith age-related cell loss, loss of mitochondrial function, or loss oftelomere function. In a still further aspect, the age-related disease ordisorder comprises an abnormal proliferative disease, a degenerativedisease, or a function-decreasing disorder. In a yet further aspect, theage-related disease or disorder is selected from tumorigenesis,malignant cancer development, myocardial infarction, cerebellarinfarction, stroke, Parkinson's disease, heart failure, atherosclerosis,hypertension, cataract, age-related macular degeneration, sarcopenia,osteoarthritis, osteoporosis, loss of bone marrow, multiple sclerosis,Sjogren, Rheumatoid arthritis, decreased immune function, diabetes,idiopathic pulmonary fibrosis, a neurodegenerating disease, Alzheimer'sdisease, Huntington's disease, and disorders caused by dysfunction intestosterone, estrogen, growth hormone, IGF-I, or energy production.

In a further aspect, the compounds and the at least one agent areco-packaged. In a still further aspect, the compounds and the at leastone agent are co-formulated. In a yet further aspect, each dose of thelithium compound and the glycyrrhizie triterpenoid compound areco-packaged. In an even further aspect, each dose of the lithiumcompound, the glycyrrhizie triterpenoid compound, and the at least oneagent are co-formulated. In a still further aspect, each dose of thelithium compound and the at least one agent are co-formulated. In a yetfurther aspect, each dose of the glycyrrhizie triterpenoid compound andthe at least one agent are co-packaged.

In a further aspect, plurality of dosage forms, the plurality comprisingone or more doses; wherein each dose comprises an effective amount ofthe lithium compound and the glycyrrhizie triterpenoid compound. In astill further aspect, each dose comprises an effective amount of thelithium compound, the glycyrrhizie triterpenoid compound, and the atleast one agent.

In a further aspect, each dose of the lithium compound and theglycyrrhizie triterpenoid compound are administered sequentially. In astill further aspect, each dose of the lithium compound and theglycyrrhizie triterpenoid compound are administered simultaneously. In ayet further aspect, each dose of the lithium compound and the at leastone agent are administered sequentially. In an even further aspect, eachdose of the glycyrrhizie triterpenoid compound and the at least oneagent are administered simultaneously. In a still further aspect, eachdose of the lithium compound, the glycyrrhizie triterpenoid compound,and the at least one agent are administered sequentially. In a yetfurther aspect, each dose of the lithium compound, the glycyrrhizietriterpenoid compound, and the at least one agent are administeredsimultaneously.

In a still further aspect, the effective amount is a therapeuticallyeffective amount. In yet a further aspect, the effective amount is aprophylactically effective amount.

In a further aspect, the dosage forms are formulated for oraladministration, inhalation, topical administration, and/or parenteraladministration. In a still further aspect, the dosage form for thecompounds is formulated for oral administration. In yet a furtheraspect, the dosage form for the compound is formulated for oraladministration and the dosage form for the at least one agent isformulated for oral administration.

In a further aspect, the agent known to treat an age-related disorder ordisease can comprise nutritional supplements, anti-inflammatorymedicines (e.g., NSAIDS) antiplatelet medicines (e.g., clopidogrel,aspirin), anticoagulants (e.g., warfarin, heparin), lipid loweringmedicines (e.g., statins, niacin), anti-hypertensive medicines (e.g.,angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptorblockers (ARBs), beta-blockers, diuretics, and calcium channel blockers(CCBs)), anti-diabetic medicines (e.g., metformin), chemotherapeuticagents, or a combination thereof. In a still further aspect, thenutritional supplement can comprise vitamins, minerals, antioxidants,amino acids, fatty acid complex, digestive enzymes, or a combinationthereof.

In a further aspect, the agent can comprise a retinoic acid compound ora pharmaceutically acceptable salt thereof. In a still further aspect,the agent can comprise all-trans-retinoic acid or a cis-retinoic acid,or pharmaceutically acceptable salts thereof, and combinations thereof.In yet further aspects, the agent is selected from all-trans-retinoicacid, 7-cis-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid,13-cis-retinoic acid, or pharmaceutically acceptable salts thereof, andcombinations thereof.

It is understood that the disclosed kits can be prepared from thedisclosed compounds, products, and pharmaceutical compositions. It isalso understood that the disclosed kits can be employed in connectionwith the disclosed methods of using.

The disclosed compositions and methods include at least the followingaspects:

Aspect 1: A method for preventing cellular aging in a cell of a subject,the method comprising the step of providing to the cell an effectiveamount of at least one lithium compound or a pharmaceutically acceptablesalt thereof, and an effective amount of at least one glycyrrhizietriterpenoid compound or a pharmaceutically acceptable salt thereof,thereby preventing the cell from aging.

Aspect 2: The method of aspect 1, wherein the least one lithiumcompound, or a pharmaceutically acceptable salt thereof is selected fromlithium chloride, lithium bromide, lithium carbonate, lithium nitrate,lithium sulfate, lithium acetate, lithium lactate, lithium citrate,lithium aspartate, lithium gluconate, lithium malate, lithium ascorbate,lithium orotate, lithium succinate, and combinations thereof.

Aspect 3: The method of aspects 1 or 2, wherein the least one lithiumcompound, or a pharmaceutically acceptable salt thereof is selected fromlithium chloride, lithium bromide, lithium carbonate, lithium citrate,and lithium orotate.

Aspect 4: The method of any preceding aspect, wherein the least oneglycyrrhizie triterpenoid compound is selected from glycyrrhizin,enoxolone, carbenoxolone, cicloxolone, pharmaceutically acceptable saltsthereof, and combinations thereof.

Aspect 5: The method of any preceding aspect, wherein the least oneglycyrrhizie triterpenoid is glycyrrhizin, having the structurerepresented by the formula:

Aspect 6: The method of any preceding aspect, wherein the least onelithium compound, or a pharmaceutically acceptable salt thereof isselected from lithium chloride, lithium bromide, lithium carbonate,lithium nitrate, lithium sulfate, lithium acetate, lithium lactate,lithium citrate, lithium aspartate, lithium gluconate, lithium malate,lithium ascorbate, lithium orotate, lithium succinate, and combinationsthereof; and wherein the least one glycyrrhizie triterpenoid compound isselected from glycyrrhizin, enoxolone, carbenoxolone, cicloxolone,pharmaceutically acceptable salts thereof, and combinations thereof.

Aspect 7: The method of any preceding aspect, wherein the least onelithium compound, or a pharmaceutically acceptable salt thereof isselected from lithium chloride, lithium bromide, lithium carbonate,lithium citrate, and lithium orotate; and wherein the least oneglycyrrhizie triterpenoid compound is selected from glycyrrhizin,enoxolone, carbenoxolone, cicloxolone, pharmaceutically acceptable saltsthereof, and combinations thereof.

Aspect 8: The method of any preceding aspect, wherein the least onelithium compound, or a pharmaceutically acceptable salt thereof isselected from lithium chloride, lithium bromide, lithium carbonate,lithium nitrate, lithium sulfate, lithium acetate, lithium lactate,lithium citrate, lithium aspartate, lithium gluconate, lithium malate,lithium ascorbate, lithium orotate, lithium succinate, and combinationsthereof; and wherein the least one glycyrrhizie triterpenoid compound isselected from glycyrrhizin, and pharmaceutically acceptable saltsthereof.

Aspect 9: The method of any preceding aspect, wherein the least onelithium compound, or a pharmaceutically acceptable salt thereof isselected from lithium chloride, lithium bromide, lithium carbonate,lithium citrate, and lithium orotate; and wherein the least oneglycyrrhizie triterpenoid compound is selected from glycyrrhizin, andpharmaceutically acceptable salts thereof.

Aspect 10: The method of any preceding aspect, wherein the least onelithium compound, or a pharmaceutically acceptable salt thereof isselected from lithium chloride, lithium bromide, lithium carbonate,lithium citrate, and lithium orotate; and wherein the least oneglycyrrhizie triterpenoid compound is selected from glycyrrhizin, andpharmaceutically acceptable salts thereof.

Aspect 11: The method of any preceding aspect, wherein the least oneglycyrrhizie triterpenoid compound is an extract from glycyrrhiza(licorice), an extract of the dried rhizome and roots of Glycyrrhizaglabra, or a combination thereof.

Aspect 12: The method of any preceding aspect, wherein the cellularanti-aging activity comprises: maintaining cell replication potential;maintaining senescence, maintaining cell cycle-arrested state inpost-mitotic cells, stimulating, improving, or maintaining mitochondrialfunction; preventing deterioration of mitochondria, preventing celldeath following senescence deterioration, or a combination thereof.

Aspect 13: The method of any preceding aspect, wherein the cellularanti-aging activity comprises: activity against at least one componentof the TOR (Target of rapamycin) pathway, IGF-I (insulin-like growthfactors) receptor pathway, EGFR (epidermal growth factor receptor)pathway, or a combination thereof.

Aspect 14: The method of any preceding aspect, wherein the cellularanti-aging activity comprises inhibition of at least one component ofthe TOR (Target of rapamycin) pathway, IGF-I (insulin-like growthfactors) receptor pathway, EGFR (epidermal growth factor receptor)pathway, or a combination thereof.

Aspect 15: The method of any preceding aspect, wherein the at least onecomponent comprises an enzyme or a protein.

Aspect 16: The method of any preceding aspect, wherein the cellularanti-aging activity comprises preventing an age-related disease ordisorder.

Aspect 17: The method of any preceding aspect, wherein the age-relateddisease or disorder is associated with age-related cell loss, loss ofmitochondrial function, or loss of telomere function.

Aspect 18: The method of any preceding aspect, wherein the age-relateddisease or disorder comprises an abnormal proliferative disease, adegenerative disease, or a function-decreasing disorder.

Aspect 19: The method of any preceding aspect, wherein the age-relateddisease or disorder is selected from tumorigenesis, malignant cancerdevelopment, myocardial infarction, cerebellar infarction, stroke,Parkinson's disease, heart failure, atherosclerosis, hypertension,cataract, age-related macular degeneration, sarcopenia, osteoarthritis,osteoporosis, loss of bone marrow, multiple sclerosis, Sjogren,Rheumatoid arthritis, decreased immune function, diabetes, idiopathicpulmonary fibrosis, a neurodegenerating disease, Alzheimer's disease,Huntington's disease, and disorders caused by dysfunction intestosterone, estrogen, growth hormone, IGF-I, or energy production.

Aspect 20: The method of any preceding aspect, wherein the cell is amammalian cell.

Aspect 21: The method of aspect 20, wherein the mammalian cell is ahuman cell.

Aspect 22: The method of any preceding aspect, wherein the effectiveamount is a therapeutically effective amount.

Aspect 23: The method of any preceding aspect, wherein the effectiveamount is a prophylactically effective amount.

Aspect 24: The method of any preceding aspect, further comprising thestep of identifying a cell in need of anti-aging treatment.

Aspect 25: The method of any preceding aspect, wherein the effectiveamount of the lithium compound and the glycyrrhizie triterpenoidcompound are from about 0.001 to about 10000 μM in serum medium.

Aspect 26: The method of any preceding aspect, wherein the effectiveamount of the lithium compound is from about 0.1 to about 10 μM in serummedium and the effective amount of the glycyrrhizie triterpenoidcompound is from about 1 to about 1000 μM in serum medium.

Aspect 27: The method of any preceding aspect, wherein the effectiveamount of the lithium compound is from about 0.1 to about 1 μM in serummedium and the effective amount of the glycyrrhizie triterpenoidcompound is from about 1 to about 100 μM in serum medium.

Aspect 28: The method of any preceding aspect, wherein the ratio oflithium compound to glycyrrhizie triterpenoid compound is from about1:10 to about 1:1000.

Aspect 29: The method of any preceding aspect, wherein the ratio oflithium compound to glycyrrhizie triterpenoid compound is from about1:50 to about 1:250.

Aspect 30: A method for preventing cellular aging activity in a subject,the method comprising the step of providing to the subject an effectiveamount of at least one intracellular alkalinizing agent or apharmaceutically acceptable salt thereof, thereby preventing thecellular aging activity.

Aspect 31: The method of aspect 30, wherein the intracellularalkalization action comprises direct competition with Na+ on asodium-hydrogen exchanger, increased expression or number ofsodium-hydrogen exchangers, intracellular Na+ retention; increasingmembrane permeability for Na+, increased Na+ reabsorption; increasedsecretion of H+, decreased accumulation of acid products of metabolism;or a combination thereof

Aspect 32: The method of any preceding aspect, further comprisingproviding to the cell an effective amount of at least one agent forimproving telomere function or a pharmaceutically acceptable saltthereof

Aspect 33: The method of any preceding aspect, wherein the step ofproviding to the subject comprises providing to the subject an effectiveamount of at least one lithium compound or a pharmaceutically acceptablesalt thereof, and an effective amount of at least one glycyrrhizietriterpenoid compound or a pharmaceutically acceptable salt thereof.

Aspect 34: The method of any preceding aspect, wherein the subject hasbeen diagnosed with a need for anti-aging treatment prior to theadministering step.

Aspect 35: The method of any preceding aspect, wherein the subject hasbeen diagnosed with an age-related disease or disorder associated withage-related cell loss, loss of mitochondrial function, or loss oftelomere function.

Aspect 36: The method of any preceding aspect, wherein the age-relateddisease or disorder comprises an abnormal proliferative disease, adegenerative disease, or a function-decreasing disorder.

Aspect 37: The method of any preceding aspect, wherein the age-relateddisease or disorder is selected from tumorigenesis, malignant cancerdevelopment, myocardial infarction, cerebellar infarction, stroke,Parkinson's disease, heart failure, atherosclerosis, hypertension,cataract, age-related macular degeneration, sarcopenia, osteoarthritis,osteoporosis, loss of bone marrow, multiple sclerosis, Sjogren,Rheumatoid arthritis, decreased immune function, diabetes, idiopathicpulmonary fibrosis, a neurodegenerating disease, Alzheimer's disease,Huntington's disease, and disorders caused by dysfunction intestosterone, estrogen, growth hormone, IGF-I, or energy production.

Aspect 38: The method of any preceding aspect, wherein the subject is amammal.

Aspect 39: The method of any preceding aspect, wherein the mammal is ahuman.

Aspect 40: The method of any preceding aspect, wherein the effectiveamount is a therapeutically effective amount.

Aspect 41: The method of any preceding aspect, wherein the effectiveamount is a prophylactically effective amount.

Aspect 42: The method of any preceding aspect, further comprising thestep of identifying a subject in need of anti-aging treatment.

Aspect 43: The method of any preceding aspect, wherein the subject inneed of anti-aging treatment comprises having at least one risk factorfor developing an age-related disease or disorder.

Aspect 44: The method of any preceding aspect, wherein the age-relateddisease or disorder is associated with age-related cell loss, loss ofmitochondrial function, or loss of telomere function.

Aspect 45: The method of any preceding aspect, wherein the age-relateddisease or disorder comprises an abnormal proliferative disease, adegenerative disease, or a function-decreasing disorder.

Aspect 46: The method of any preceding aspect, wherein the age-relateddisease or disorder is selected from tumorigenesis, malignant cancerdevelopment, myocardial infarction, cerebellar infarction, stroke,Parkinson's disease, heart failure, atherosclerosis, hypertension,cataract, age-related macular degeneration, sarcopenia, osteoarthritis,osteoporosis, loss of bone marrow, multiple sclerosis, Sjogren,Rheumatoid arthritis, decreased immune function, diabetes, idiopathicpulmonary fibrosis, a neurodegenerating disease, Alzheimer's disease,Huntington's disease, and disorders caused by dysfunction intestosterone, estrogen, growth hormone, IGF-I, or energy production.

Aspect 47: The method of any preceding aspect, further comprising thestep of providing to the cell an effective amount of at least oneretinoic acid compound or a pharmaceutically acceptable salt thereof.

Aspect 48: The method of any preceding aspect, wherein the at least oneretinoic acid compound is selected from all-trans-retinoic7-cis-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, and13-cis-retinoic acid.

Aspect 49: The method of any preceding aspect, wherein the effectiveamount of the retinoic acid compound is from about 0.001 to about 10000μM in serum medium.

Aspect 50: A method for the treatment of a subject, the methodcomprising the steps of: (a) diagnosing the subject as having anage-related disorder or disease; and (b) administering to the subject aneffective amount of at least one lithium compound or a pharmaceuticallyacceptable salt thereof, and an effective amount of at least oneglycyrrhizie triterpenoid compound or a pharmaceutically acceptable saltthereof.

Aspect 51: The method of any preceding aspect, wherein diagnosingcomprises performing a physical examination upon the subject and makinga finding.

Aspect 52: The method of any preceding aspect, wherein the findingcomprises identifying at least one risk factor for developing anage-related disease or disorder.

Aspect 53: The method of any preceding aspect, wherein diagnosingcomprises performing an experiment upon the subject and identifying alevel of a biological marker.

Aspect 54: The method of any preceding aspect, wherein the biologicalmarker is a marker for an age-related disease or disorder.

Aspect 55: The method of any preceding aspect, where in the subject is abiological sample.

Aspect 56: The method of any preceding aspect, where in the biologicalsample is selected from a cell, blood, saliva, urine, tissue, or phlegm.

Aspect 57: The method of any preceding aspect, wherein the age-relateddisease or disorder is associated with age-related cell loss, loss ofmitochondrial function, or loss of telomere function.

Aspect 58: The method of any preceding aspect, wherein the age-relateddisease or disorder comprises an abnormal proliferative disease, adegenerative disease, or a function-decreasing disorder.

Aspect 59: The method of any preceding aspect, wherein the age-relateddisease or disorder is selected from tumorigenesis, malignant cancerdevelopment, myocardial infarction, cerebellar infarction, stroke,Parkinson's disease, heart failure, atherosclerosis, hypertension,cataract, age-related macular degeneration, sarcopenia, osteoarthritis,osteoporosis, loss of bone marrow, multiple sclerosis, Sjogren,Rheumatoid arthritis, decreased immune function, diabetes, idiopathicpulmonary fibrosis, a neurodegenerating disease, Alzheimer's disease,Huntington's disease, and disorders caused by dysfunction intestosterone, estrogen, growth hormone, IGF-I, or energy production.

Aspect 60: The method of any preceding aspect, wherein the effectiveamount of the lithium compound is from about 1 mg to about 50 mg and theeffective amount of the glycyrrhizie triterpenoid compound is from about1 to about 100 mg.

Aspect 61: The method of any preceding aspect, wherein the effectiveamount of the lithium compound is from about 1 to about 25 mg and theeffective amount of the glycyrrhizie triterpenoid compound is from about1 to about 50 mg.

Aspect 62: The method of any preceding aspect, further comprisingadministrating to the subject an effective amount of at least oneretinoic acid compound or a pharmaceutically acceptable salt thereof.

Aspect 63: The method of any preceding aspect, wherein the effectiveamount of the retinoic acid compound is from about 1 to about 100 mg.

Aspect 64: A pharmaceutical composition comprising an effective amountof at least one lithium compound or a pharmaceutically acceptable saltthereof; and an effective amount of at least one glycyrrhizietriterpenoid compound or a pharmaceutically acceptable salt thereof.

Aspect 65: The composition of any preceding aspect, wherein theeffective amount is a therapeutically effective amount.

Aspect 66: The composition of any preceding aspect, wherein theeffective amount is a prophylactically effective amount.

Aspect 67: The composition of any preceding aspect, wherein thecomposition is formulated for oral administration.

Aspect 68: The composition of any preceding aspect, wherein thecomposition is formulated for topical administration.

Aspect 69: A nutraceutical composition comprising an effective amount ofat least one lithium compound or a pharmaceutically acceptable saltthereof; and an effective amount of at least one glycyrrhizietriterpenoid compound or a pharmaceutically acceptable salt thereof.

Aspect 70: The composition of any preceding aspect, further comprisingan effective amount of at least one retinoic acid compound or apharmaceutically acceptable salt thereof.

Aspect 71: A kit comprising an effective amount of at least one lithiumcompound or a pharmaceutically acceptable salt thereof, an effectiveamount of at least one glycyrrhizie triterpenoid compound or apharmaceutically acceptable salt thereof, and one or more of: (a) atleast one agent known to treat an age-related disorder or disease; (b)instructions for treating the age-related disorder or disease; and (c)instructions for administering the lithium compound and the glycyrrhizietriterpenoid compound in connection with the age-related disorder ordisease.

Aspect 72: The kit of aspect 71, wherein each dose of the lithiumcompound and the glycyrrhizie triterpenoid compound are co-packaged.

Aspect 73: The kit of any preceding aspect, wherein each dose of thelithium compound, the glycyrrhizie triterpenoid compound, and the atleast one agent are co-formulated.

Aspect 74: The kit of any preceding aspect, wherein each dose of thelithium compound and the at least one agent are co-formulated.

Aspect 75: The kit of any preceding aspect, wherein each dose of theglycyrrhizie triterpenoid compound and the at least one agent areco-packaged.

Aspect 76: The kit of any preceding aspect, further comprising aplurality of dosage forms, the plurality comprising one or more doses;wherein each dose comprises an effective amount of the lithium compoundand the glycyrrhizie triterpenoid compound.

Aspect 77: The kit of any preceding aspect, further comprising aplurality of dosage forms, the plurality comprising one or more doses;wherein each dose comprises an effective amount of the lithium compound,the glycyrrhizie triterpenoid compound, and the at least one agent.

Aspect 78: The kit of any preceding aspect, wherein the effective amountis a therapeutically effective amount.

Aspect 79: The kit of any preceding aspect, wherein the effective amountis a prophylactically effective amount.

Aspect 80: The kit of any preceding aspect, wherein each dose of thelithium compound and the glycyrrhizie triterpenoid compound areadministered sequentially.

Aspect 81: The kit of any preceding aspect, wherein each dose of thelithium compound and the glycyrrhizie triterpenoid compound areadministered simultaneously.

Aspect 82: The kit of any preceding aspect, wherein each dose of thelithium compound and the at least one agent are administeredsequentially.

Aspect 83: The kit of any preceding aspect, wherein each dose of theglycyrrhizie triterpenoid compound and the at least one agent areadministered simultaneously.

Aspect 84: The kit of any preceding aspect, wherein each dose of thelithium compound, the glycyrrhizie triterpenoid compound, and the atleast one agent are administered sequentially.

Aspect 85: The kit of any preceding aspect, wherein each dose of thelithium compound, the glycyrrhizie triterpenoid compound, and the atleast one agent are administered simultaneously.

Aspect 86: The kit of any preceding aspect, wherein the dosage forms areformulated for oral administration, inhalation administration, topicaladministration, and/or parenteral administration.

Aspect 87: The kit of any preceding aspect, wherein the age-relateddisorder or disease is associated with age-related cell loss, loss ofmitochondrial function, or loss of telomere function.

Aspect 88: The kit of any preceding aspect, wherein the age-relateddisease or disorder comprises an abnormal proliferative disease, adegenerative disease, or a function-decreasing disorder.

Aspect 89: The kit of any preceding aspect, wherein the age-relateddisease or disorder is selected from tumorigenesis, malignant cancerdevelopment, myocardial infarction, cerebellar infarction, stroke,Parkinson's disease, heart failure, atherosclerosis, hypertension,cataract, age-related macular degeneration, sarcopenia, osteoarthritis,osteoporosis, loss of bone marrow, multiple sclerosis, Sjogren,Rheumatoid arthritis, decreased immune function, diabetes, idiopathicpulmonary fibrosis, a neurodegenerating disease, Alzheimer's disease,Huntington's disease, and disorders caused by dysfunction intestosterone, estrogen, growth hormone, IGF-I, or energy production.

Aspect 90: The kit of any preceding aspect, wherein the agent known totreat an age-related disorder or disease comprises nutritionalsupplements, anti-inflammatory medicines, antiplatelet medicines,anticoagulants, lipid lowering medicines, anti-hypertensive medicines,anti-diabetic medicines, chemotherapeutic agents, or a combinationthereof.

Aspect 91: The kit of any preceding aspect, wherein the nutritionalsupplement comprises vitamins, minerals, antioxidants, amino acids,fatty acid complex, digestive enzymes, or a combination thereof.

Aspect 92: The kit of any preceding aspect, further comprising aneffective amount of at least one retinoic acid compound or apharmaceutically acceptable salt thereof, and instructions foradministering the retinoic acid compound in connection with theage-related disorder or disease.

F. EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

The Examples are provided herein to illustrate the invention, and shouldnot be construed as limiting the invention in any way. Examples areprovided herein to illustrate the invention and should not be construedas limiting the invention in any way.

1. Beta-Galactosidase Staining of Normal Human Fibroblasts

Aging-associated beta-galactosidase is widely used as a biomarker ofsenescence. Here we investigatedsenescence-associated-beta-galactosidase activity of human colonfibroblast cells by visual assessment.

In this Example, the effect of treatment with lithium and glycyrrhizicacid separately, and as a fixed combination, was evaluated on normalhuman colon fibroblast cell line CCD 841 CoN (CRL-1790, ATCC).

The general study methods of study were as follows:

Flasks, Thermo Scientific Nunclon Delta Surface 12.5 cm (Cat. No 136196)with EMEM complete medium were seeded with cell culture of CCD 841 CoN(CRL-1790, ATCC) and were kept at 37° C.; 5% CO2 in incubator until 100%confluence was reached (2 days). Next, the flasks were kept in CO2incubator, at 37 degrees and 5% of CO2 for additional 4 days to age thecells. After the 4 days, the EMEM medium in the flask were changed tothe same medium with or without lithium carbonate and glycyrrhizic acidas explained below:

Group 1 (flasks 1-3), the medium was changed to complete EMEM mediumwith 0.15 μM lithium carbonate (Li₂CO₃; Sigma-Aldrich; Cat. No. 62472).

Group 2 (flasks 4-6), the medium was changed to complete EMEM mediumwith 0.25 mM glycyrrhizic acid (Glycyrrhizic acid ammonium salt fromammonium salt root (licorice); Sigma-Aldrich; Cat. No. G2137).

Group 3 (flasks 7-9), the medium was changed to complete EMEM mediumwith both 0.15 μM lithium carbonate and 0.25 mM glycyrrhizic acid (0.25mM).

Group 4 (flasks 10-12, control), the medium was changed to fresh competeEMEM medium without drugs as controls.

All flasks were kept in the CO₂ incubator 5% CO₂, 37° C. for additional48 hours for treatment. After the treatment period, all flasks werestained using senescence beta-galactosidase staining kit cell signaling(Cat. No. 9860) according to the protocol provided by the kit manual.

After staining, the flasks were examined by light microscope under 100×magnification 100× and visually evaluated and scored for visiblepositive beta-Galactosidase staining. FIGS. 1-4 show pictures of thestaining results for each of the groups, respectively.

As seen in FIGS. 1 and 2, the lithium carbonate treated fibroblasts andglycyrrhizic acid treated fibroblasts both stained positively forbeta-galactosidase. Additionally, as seen in FIG. 4, the control(non-treated) fibroblasts also stained positively forbeta-galactosidase. However, as seen in FIG. 3, fibroblasts treatedusing the combination of lithium carbonate and glycyrrhizic acid showedalmost no positive stain for beta-galactosidase.

Digital pictures showing the staining results for each of the 12 flaskswere then independently examined and scored for percentage of area ofgreen (positive) staining based on pixels, with and without backgroundsubtraction. The data for this evaluation is provided in Tables 1 and 2.

TABLE 1 Total original Green stained % green Image # area (pixels²) area(pixels²) staining 3357 17280000 141144.5 .82 3359 17280000 212050.51.23 3362 17280000 226344.5 1.31 3370 17280000 1147244 6.64 337517280000 959792.5 5.55 3378 17280000 912119 5.28 3386 17280000 488604.52.83 3388 17280000 1080455 6.25 3390 17280000 905959 5.24 3395 172800001193624 6.91 3396 17280000 1573116 9.1 3397 17280000 1733095.5 10.03

TABLE 2 Background Selected green Average % green Image # subtractionarea green area stained 3357 w/o 137278 141144.5 0.816808449 w 1450113359 w/o 173646 212050.5 1.227144097 w 250455 3362 w/o 229830 226344.51.309864005 w 222859 3370 w/o 1236353 1147243.5 6.639140625 w 10581343375 w/o 845048 959792.5 5.554354745 w 1074537 3378 w/o 825052 9121195.278466435 w 999186 3386 w/o 362321 488604.5 2.827572338 w 614888 3388w/o 860394 1080455 6.252633102 w 1300516 3390 w/o 786299 9059595.242818287 w 1025619 3395 w/o 1287179 1193624 6.907546296 w 11000693396 w/o 1597579 1573116 9.103680556 w 1548653 3397 w/o 16845701733095.5 10.02948785 w 1781621

Table 3 shows the data from analyzed pictures for the correspondingflask and FIG. 5 shows the average % of green staining for each group.Higher values reflect more intensive green staining which corresponds tomore aged cells.

TABLE 3 Flask # Area % green staining 1 control 3370 6.64 2 control 33755.55 3 control 3378 5.28 4 G acid 3396 9.1 5 G acid 3397 10.03 6 G acid3395 6.91 7 Li 3388 6.25 8 Li 3386 2.83 9 Li 3390 5.24 10 Li and G 33570.82 acid 11 Li and G 3362 1.31 acid 12 Li and G 3359 1.23 acid

As the data show, lithium carbonate and glycyrrhizic acid alone did notappear to have significant effect on beta-galactosidase staining of thefibroblasts in comparison with non-treated controls.

However, cells treated with the combination of lithium and glycyrrhizicacid exhibited significantly less positive staining forbeta-galactosidase in comparison with the lithium monotherapy andglycyrrhizic acid monotherapy. The data suggest the lithium andglycyrrhizic acid combination have a rejuvenation or anti-aging effecton the cells. Surprisingly, this rejuvenation effect is visiblyobservable in combination therapy, but is relevantly undetectable withmonotherapy treatment.

2. Beta-Galactosidase Staining Of Normal Human Fibroblasts

In this second Example, the effect of treatment with lithium andglycyrrhizic acid separately, and as a fixed combination, was furtherevaluated on normal human colon fibroblast cell line CCD 841 CoN(CRL-1790, ATCC).

The general study methods of study were as follows:

40 flasks Thermo Scientific Nunc Nunclon Delta Surface 12.5 cm (Cat. No.136196) with EMEM complete medium were seeded with cell culture of CCD841 CoN (CRL-1790, ATCC) and were kept at 37° C.; 5% CO₂ in incubatoruntil 100% confluence was reached (2 days). Next, the flasks were keptin CO2 incubator, at 37 degrees and 5% of CO2 for additional 4 days toage the cells. After the 4 days, the EMEM medium in the flask werechanged to the same medium with or without lithium carbonate andglycyrrhizic acid as explained below:

Group #1 contained complete EMEM medium with lithium carbonate 0.15 μM.

Group #2 contained complete EMEM medium with lithium carbonate 1.5 μM.

Group #3 contained complete EMEM medium with lithium carbonate 15 μM.

Group #4 contained complete EMEM medium with lithium carbonate 150 μM.

Group #5 contained complete EMEM medium with lithium carbonate 0.15 μMand glycyrrhizic acid 0.25 mM.

Group #6 contained complete EMEM medium with lithium carbonate 1.5 μMand glycyrrhizic acid 0.25 mM.

Group#7 contained complete EMEM medium with lithium carbonate 15 μM andglycyrrhizic acid 0.25 mM.

Group #8 contained complete EMEM medium with lithium carbonate 150 μMand glycyrrhizic acid 0.25 mM.

Group #9 contained complete EMEM medium with glycyrrhizic acid 0.25 mM.

Group #10 contained complete EMEM medium only and was used as a control.

All flasks were kept in the CO₂ incubator 5% CO2, 37° C. for additional48 hours for treatment. After the treatment period, all flasks werestained using senescence beta-galactosidase staining kit cell signalingaccording to the protocol provided by the kit manual.

After staining, the flasks were examined by light microscope under 100×magnification 100× and visually evaluated and scored for visiblepositive beta-Galactosidase staining. FIGS. 6-15 show pictures of thestaining results for each of the groups, respectively. As the datashows, lithium carbonate 15 μM and glycyrrhizic acid 0.25 mMcombotherapy (Group #7) showed minimal green staining, which correspondsto maximum rejuvenation effect.

3. Beta-Galactosidase Staining of Normal Human Fibroblasts

In this third Example, the effect of treatment with lithium andglycyrrhizic acid separately, and as a fixed combination, was furtherevaluated on normal human colon fibroblast cell line CCD 841 CoN(CRL-1790, ATCC).

The general study methods of study were as follows:

Flasks Thermo Scientific Nunc Nunclon Delta Surface 12.5 cm with EMEMcomplete medium were seeded with cell culture of CCD 841 CoN (CRL-1790,ATCC) and were kept at 37° C.; 5% CO₂ in incubator until 100% confluencewas reached (2 days). Next, the flasks were kept in CO2 incubator, at 37degrees and 5% of CO2 for additional 4 days to age the cells. After the4 days, the EMEM medium in the flask were changed to the same mediumwith or without lithium carbonate and/or glycyrrhizic acid as explainedbelow:

Group #1 contained complete EMEM medium with lithium carbonate in finalconcentration of 0.15 μM.

Group #2 contained complete EMEM medium with lithium carbonate in finalconcentration of 1.5 μM.

Group #3 contained complete EMEM medium with lithium carbonate in finalconcentration of 15 μM.

Group #4 contained complete EMEM medium with lithium carbonate in finalconcentration of 150 μM.

Group #5 contained complete EMEM medium with lithium carbonate in finalconcentration of 0.15 μM and glycyrrhizic acid in final concentration of0.25 mM.

Group #6 contained complete EMEM medium with lithium carbonate in finalconcentration of 1.5 μM and Glycyrrhizic acid in final concentration of0.25 mM.

Group #7 contained complete EMEM medium with lithium carbonate in finalconcentration of 15 μM and Glycyrrhizic acid in final concentration of0.25 mM.

Group #8 contained complete EMEM medium with lithium carbonate in finalconcentration of 150 μM and glycyrrhizic acid in final concentration of0.25 mM.

Group #9 contained complete EMEM medium with glycyrrhizic acid in finalconcentration of 0.25 mM.

Group #10 contained complete EMEM medium only and was used as a control.

All flasks were kept in the CO₂ incubator 5% CO₂, 37° C. for additional48 hours for treatment. After the treatment period, the cells were thendetached from growth surface and collected in Eppendorf PCR-clean tubedin volume of 100 μL and dissolved in 0,5 ml of TRI reagent. Next, qPCRwas performed in order to evaluate gene expression profile of the cellin control and treatments groups. The data from the qPCR studies areprovided in FIG. 16.

As the data shows, combination therapy using glycyrrhizic acid andlithium carbonate produced diminished IGF-1, while concurrentlyincreasing telomerase RNA component. Moreover, the specific combinationof 0.25 mM glycyrrhizic acid with 15 μM lithium carbonate also appearsto decrease IGF-1R and EGFR gene expression levels.

4. C. Elegans Longevity Study

In this fourth Example, the effect of treatment with lithium andGlycyrrhizic acid separately, and as a fixed combination, was furtherevaluated on the wild-type N2 Bristol strain of C. elegans. Worm stockswere maintained on nematode growth medium (NGM) dishes containing 10μg/mL cholesterol (Cat. No. C8667; Sigma-Aldrich, Saint Louis, Mo.,USA). Dishes were seeded with OP50 Escherichia coli as a food source.

The general study methods of study were as follows:

Twelve polystyrene petri dishes (35 mm diameter) were prepared byfilling with NGM agar containing 10 mg/ml cholesterol, and 25 μM5-Fluoro-2′-deoxyuridine to suppress reproduction (FUDR; Cat. No. F0503;Sigma-Aldrich, Saint Louis, Mo., USA). The dishes were seeded with OP50Escherichia coli as a food source. Duplicate dishes were used for eachgroup.

The treatment drug was dissolved in deionized water, and added to thedishes for the groups as explained below:

Group #1: lithium carbonate in concentration of 0.15 μM.

Group #2: lithium carbonate in concentration of 15 μM.

Group #3: lithium carbonate in concentration of 0.15 μM and Glycyrrhizicacid in concentration of 0.25 mM.

Group #4: lithium carbonate in concentration of 15 μM and Glycyrrhizicacid in concentration of 0.25 mM.

Group #5: Glycyrrhizic acid in concentration of 0.25 mM.

Group #6: untreated (control).

NGM-agar treatment plates containing cholesterol (10 μg/mL) and 25 μM5-Fluoro-2′-deoxyuridine (FUDR; Cat. No. F0503; Sigma-Aldrich, SaintLouis, Mo., USA) to suppress reproduction.

Treatment plates were grown overnight at room temperature (20±2° C.),and synchronized young adult worms were added to achieve a density of19-66 worms per plate. The worms were monitored seven times a week formortality and scored as dead when they failed to respond when proddedwith a platinum pick. The data for this study is provided in Tables 4and 5 below. The survival data for the various groups are also depictedin FIG. 17.

TABLE 4 Restricted mean Age in days at % mortality No. of Std. 95% 95%Median Name subjects Days error C.I. 25% 50% 75% 90% 100% C.I. Control66 15.53 0.21 15.11~15.95 15 16 17 18 20 15~15 Ga 85 16.06 0.2715.53~16.59 14 16 18 19 20 15~16 Li 15 uM 57 14.56 0.25 14.06~15.06 1415 16 18 19 14~14 Li 0.15 uM 66 14.88 0.26 14.37~15.39 13 15 16 18 1914~15 li 15 uMGa 41 15.49 0.43 14.65~16.32 14 16 17 18 — 15~15 li 0.15Ga 132 17.65 0.22 17.22~18.08 16 18 20 21 — 17~18

TABLE 5 Statistics Condition Chi{circumflex over ( )}2 P-valueBonferroni P-value Control v.s. Ga 6.06 0.0139 0.0693 Control v.s. Li 15uM 6.42 0.0113 0.0563 Control v.s. Li0.15 uM 4.52 0.0335 0.1673 Controlv.s. li15 uM Ga 1.03 0.3106 1.0000 Control v.s. li0.15 Ga 46.70 0.0e+000.0e+00 Ga v.s. Control 6.06 0.0139 0.0693 Ga v.s. Li 15 uM 17.812.4e−05 0.0001 Ga v.s. Li0.15 uM 11.51 0.0007 0.0035 Ga v.s. li15 uM Ga1.51 0.2191 1.0000 Ga v.s. li0.15 Ga 25.35 4.8e−07 2.4e−06 Li 15 uM v.s.Control 6.42 0.0113 0.0563 Li 15 uM v.s. Ga 17.81 2.4e−05 0.0001 Li 15uM v.s. Li0.15 uM 1.05 0.3051 1.0000 Li 15 uM v.s. li15 uM Ga 7.340.0067 0.0336 Li 15 uM v.s. li0.15 Ga 72.06 0.0e+00 0.0e+00 Li0.15 uMv.s. Control 4.52 0.0335 0.1673 Li0.15 uM v.s. Ga 11.51 0.0007 0.0035Li0.15 uM v.s. Li 15 uM 1.05 0.3051 1.0000 Li0.15 uM v.s. li15 uM Ga10.03 0.0015 0.0077 Li0.15 uM v.s. li0.15 Ga 78.35 0.0e+00 0.0e+00 li15uM Ga v.s. Control 1.03 0.3106 1.0000 li15 uM Ga v.s. Ga 1.51 0.21911.0000 li15 uM Ga v.s. Li 15 uM 7.34 0.0067 0.0336 li15 uM Ga v.s.Li0.15 uM 10.03 0.0015 0.0077 li15 uM Ga v.s. li0.15 Ga 22.77 1.8e−069.1e−06 li0.15 Ga v.s. Control 46.70 0.0e+00 0.0e+00 li0.15 Ga v.s. Ga25.35 4.8e−07 2.4e−06 li0.15 Ga v.s. Li 15 uM 72.06 0.0e+00 0.0e+00li0.15 Ga v.s. Li0.15 uM 78.35 0.0e+00 0.0e+00 li0.15 Ga v.s. li15 uM Ga22.77 1.8e−06 9.1e−06

As the data show, combination therapy using 0.15 uM lithium carbonatewith 0.25 mM glycyrrhizic acid significantly increased longevity andsurvival of the wild type C. elegans specimens. Surprisingly, thelongevity effect is observed with specific concentrations of lithiumcarbonate and glycyrrhizic acid combination therapy, and is not observedat higher combination therapy concentrations or with lithium carbonatemonotherapy and glycyrrhizic acid monotherapy.

5. Beta-Galactosidase Staining of Normal Human Fibroblasts

In this next Example, the effect of treatment with lithium, glycyrrhizicacid, and retinoic acid, in various combinations, was further evaluatedon normal human colon fibroblast cell line CRL-1790 (ATCC).

The general study methods were as follows:

4 flasks (Thermo Scientific Nunc Nunclon Delta Surface 12.5 cm; Cat. No.136196) with McCoy's 5A complete medium were seeded with cell culture ofCRL1790 (ATCC) and were kept at 37° C.; 5% CO₂ in incubator until 100%confluence was reached (2 days). After reaching full confluence, inorder to age cells, the flasks were kept in CO2 incubator, at 37 degreesand 5% of CO2 for an additional 10 days. After 10 days of aging, theMcCoy's 5A medium with 10% of FBS was changed to the same medium with orwithout lithium carbonate and glycyrrhizic acid and the same combinationwith the addition of 1 uM of retinoic acid as explained below:

Flask 1: medium was changed to complete McCoy's 5A medium with LithiumCarbonate Li2CO3 (Sigma-Aldrich; Cat. #62472) in concentration of 0.15uM and glycyrrhzic acid (Glycyrrhizic acid ammonium salt from ammoniumsalt root (licorice); Sigma-Aldrich; Cat. # G2137) in concentration of0.25 mM.

Flask 2: medium was changed to complete McCoy's SA medium with LithiumCarbonate (0.15 uM) and Glycyrrhzic acid (0.25 mM) and retinoic acid (1uM) (Sigma-Aldrich; Cat # R2625).

Flask 3: medium was changed to complete McCoy's 5A medium with retinoicacid (1 uM) only.

Flask 4: medium was changed to fresh McCoy's 5A compete medium only. Allflasks were kept in the CO₂ incubator 5% CO2, 37° C. for additional 24hours for treatment. After the treatment period, all flasks were stainedusing senescence beta-galactosidase staining kit cell signalingaccording to the protocol provided by the kit manual.

After staining, the flasks were examined by light microscope under 100×magnification 100× and visually evaluated and scored for visiblepositive beta-Galactosidase staining. FIG. 18 shows a picture of thestaining results for the control group (non-treated fibroblasts stainedpositively for beta-Galactosidase). FIG. 19 shows a picture of thestaining results for the retinoic acid mono-treated fibroblasts stainedpositively for beta-galactosidase. FIG. 20 shows a picture of thestaining results for the fibroblasts treated with a combination oflithium carbonate, glycyrrhizic acid, retinoic acid, and stainedpositively for beta-galactosidase. FIG. 21 shows a picture of thestaining results for the lithium carbonate and glycyrrhizic acid treatedfibroblasts stained positively for beta-galactosidase.

As the data show, the groups treated with both of lithium andglycyrrhizic acid show significantly less positive staining forbeta-galactosidases in comparison with the non-treated control. Thiseffect was more prominent when adding of 1 uM of retinoic acid. Theretinoic acid monotherapy flask did not show significant difference fromthe control group.

6. qPCR Measurement of Telomere Length and Differential Gene Expressionin a Human Volunteer

In this next Example, the effect of treatment with lithium orotate andlicorice root extract was further evaluated on relative telomere lengthand the mRNA expression of telomere associated genes—telomerase RNAcomponent (TERC) and dyskerin—in the blood cells of healthy humanvolunteer.

The general study methods were as follows:

DNA and RNA was extracted using QiaAmp DNA mini kit (Qiagen) accordingto manufacturer's instructions. DNA samples were used to assess therelative telomere length by qPCR using previously published method(Cawthon, Nucleic Acid Research, 2002). cDNA samples were used to assessmRNA expression PF markers, using qPCR, with FastStart Universal SYBRGreen Master with Rox (Roche) and StepOnePlus Real-Time PCR System(Applied Biosystems). Telomere length was normalized to single copy DNA36B4 and gene expression was normalized to the house-keeping genes HPRT.All data were analyzed using comparative 2−^(ΔΔCt) method. Table 6 showsthe primer sets that were used:

TABLE 6 Telomere h-Tel  CGGTTTGTTTGGGTTTGGGT length (F)TTGGGTTTGGGTTTGGGTT h-Tel  GGCTTGCCTTACCCTTACCC (R) TTACCCTTACCCTTACCCTDyskerin DKC1 AAAGACCGGAAGCCATTACA telomers (DKC1) (F) AG DKC1GCCACTGAGAAGTGTCTAAT telomers (R) TGA Telomerase TERC CCGCTGTTTTTCTCGCTGAC telomers RNA (F) component (TERC)

FIG. 22 summarizes data obtained following real time analysis of bloodcells from the healthy human volunteer. FIG. 23 shows a heatmapoutlining the effect of inhaled lithium on telomere lengthening. As thedata shows, administration of lithium/licorice combination increased theaverage telomere length by 3-fold in comparison to the same individualbaseline. Moreover, the level of telomerase components, known to play aphysiological role in the 3′ elongation of telomeres via addition ofTTAGGG—TERC and dyskerin—were elevated as well. As shown in FIG. 23, anincrease in TERC and dyskerin was observed with contrasting reduction inthe expression of fibrosis-associated genes.

7. Gene Expression of Aging Biomarkers

In this next Example, the effect of treatment with lithium orotate andglyccyrhizic acid was further evaluated on 8 signature biomarkers (4upregulated and 4 downregulated) of aging, as identified in leadingresearch groups in the field (Magalhaes et al, Gene Expression, 2009).

The genes overexpressed by aging examined were:

Alipoprotein D (APOD)—is associated with neurological disorders andnerve injury.

Fc fragment of IgG, low affinity II b (FCGR2B)—ligation to B cellsdownregulates antibody production and may, in some circumstances,promote apoptosis.

Complement component 3 (C3)—plays a central role in the activation ofthe complement system.

Clusterin (CLU)—a chaperone associated with the clearance of cellulardebris and apoptosis.

The genes suppressed by aging examined were:

Transferrin receptor (TFRC)—is required for iron import from transferrininto cells by endocytosis.

Collagen type III (COL3)—found in found in extensible connective tissuessuch as skin, lung, and the vascular system.

ATP synthase (ATP5G3)—generates energy in the mitochondria (ATP fromADP).

NADH dehydrogenase (NDUFB11)—participates in mitochondrial oxidativephosphorylation.

The general study methods were as follows:

(Thermo Scientific Nunc Nunclon Delta Surface 12.5 cm; Cat. No. 136196)with McCoy's 5A complete medium were seeded with cell culture of CRL1790(ATCC) and were kept at 37° C.; 5% CO₂ in incubator until 100%confluence was reached (2 days).

To 80 cm² flasks with CRL-1790 cells in confluence about 50-60%, McCoy'smedium with 10% of FCS lithium carbonate (SigmaAldrich Cat #62472) wasadded in order to establish concentration of 10 μM, for 48 hours. Cellsfrom both flasks, treated by lithium carbonate alone, glyccyrhizic acidalone and combination of both and control were detached by exposition to5 ml of Trypsin EDTA. Flasks were then placed to incubate at 37° C.,5%-CO2 for 10 min and collected to 15 ml sterile tubes. Next, the cellswere centrifuged at 600 rpm for 10 minutes; supernatant discarded and0.1 ml pellet of density 2×10⁶ cells/ml was dissolved in 0.5 ml of Trireagent. Total RNA was extracted using TRI-reagent PureLink RNA Mini Kit(Life Technologies) and reverse transcribed using PrimeScript RT MasterMix (Clontech). cDNA was diluted 1:25 and used for real-time qPCR withFastStart Universal SYBR Green Master with Rox (Roche) and StepOnePlusReal-Time PCR System (Applied Biosystems). FIG. 24 shows the results ofthe real-time PCR.

As the data show, in CRL179 cells, 0.25 mM glyccyrhizic acid+0.15 μMlithium exhibited the most robust effect on Complement C3 andClusterin—significantly reducing their levels, which are regularlyelevated with aging. Increased levels of ATP synthase and NADHdehydrogenase were also observed, which are regularly reduced by aging.Other genes did not show significantly altered expression. The datasuggests that 0.25 mM glyccyrhizic acid+0.15 μM lithium contributed torejuvenation of cells, boosting their mitochondrial activity andsuppressing inflammation. Without wishing to be bound by a particulartheory, it is believed that lithium and the active component of licoricealters molecular profile, shifting cells toward a more youthful state.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otheraspects of the invention will be apparent to those skilled in the artfrom consideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

1. A method for preventing cellular aging in a cell of a subject, themethod comprising the step of providing to the cell an effective amountof at least one lithium compound or a pharmaceutically acceptable saltthereof, and an effective amount of at least one glycyrrhizictriterpenoid compound or a pharmaceutically acceptable salt thereof,thereby preventing the cell from aging.
 2. The method of claim 1,wherein the least one lithium compound, or a pharmaceutically acceptablesalt thereof is selected from lithium chloride, lithium bromide, lithiumcarbonate, lithium nitrate, lithium sulfate, lithium acetate, lithiumlactate, lithium citrate, lithium aspartate, lithium gluconate, lithiummalate, lithium ascorbate, lithium orotate, lithium succinate, andcombinations thereof.
 3. (canceled)
 4. The method of claim 1, whereinthe least one glycyrrhizic triterpenoid compound is selected fromglycyrrhizin, enoxolone, carbenoxolone, cicloxolone, pharmaceuticallyacceptable salts thereof, and combinations thereof.
 5. The method ofclaim 4, wherein the least one glycyrrhizic triterpenoid isglycyrrhizin, having the structure represented by the formula:

6.-11. (canceled)
 12. The method of claim 1, wherein the cellularanti-aging activity comprises: maintaining cell replication potential;maintaining senescence, maintaining cell cycle-arrested state inpost-mitotic cells, stimulating, improving, or maintaining mitochondrialfunction; preventing deterioration of mitochondria, preventing celldeath following senescence deterioration, or a combination thereof. 13.The method of claim 1, wherein the cellular anti-aging activitycomprises: activity against at least one component of the TOR (Target ofrapamycin) pathway, IGF-I (insulin-like growth factors) receptorpathway, EGFR (epidermal growth factor receptor) pathway, or acombination thereof.
 14. The method of claim 13, wherein the cellularanti-aging activity comprises inhibition of at least one component ofthe TOR (Target of rapamycin) pathway, IGF-I (insulin-like growthfactors) receptor pathway, EGFR (epidermal growth factor receptor)pathway, or a combination thereof.
 15. (canceled)
 16. The method ofclaim 1, wherein the cellular anti-aging activity comprises preventingan age-related disease or disorder.
 17. The method of claim 16, whereinthe age-related disease or disorder is associated with age-related cellloss, loss of mitochondrial function, or loss of telomere function. 18.The method of claim 16, wherein the age-related disease or disordercomprises an abnormal proliferative disease, a degenerative disease, ora function-decreasing disorder.
 19. The method of claim 16, wherein theage-related disease or disorder is selected from tumorigenesis,malignant cancer development, myocardial infarction, cerebellarinfarction, stroke, Parkinson's disease, heart failure, atherosclerosis,hypertension, cataract, age-related macular degeneration, sarcopenia,osteoarthritis, osteoporosis, loss of bone marrow, multiple sclerosis,Sjogren, Rheumatoid arthritis, decreased immune function, diabetes,idiopathic pulmonary fibrosis, a neurodegenerating disease, Alzheimer'sdisease, Huntington's disease, and disorders caused by dysfunction intestosterone, estrogen, growth hormone, IGF-I, or energy production.20.-46. (canceled)
 47. The method of claim 1, further comprising thestep of providing to the cell an effective amount of at least oneretinoic acid compound or a pharmaceutically acceptable salt thereof.48. The method of claim 47, wherein the at least one retinoic acidcompound is selected from all-trans-retinoic 7-cis-retinoic acid,9-cis-retinoic acid, 11-cis-retinoic acid, and 13-cis-retinoic acid. 49.(canceled).
 50. A method for the treatment of a subject, the methodcomprising the steps of: a. diagnosing the subject as having anage-related disorder or disease; and b. administering to the subject aneffective amount of at least one lithium compound or a pharmaceuticallyacceptable salt thereof, and an effective amount of at least oneglycyrrhizic triterpenoid compound or a pharmaceutically acceptable saltthereof. 51.-56. (canceled)
 57. The method of claim 50, wherein theage-related disease or disorder is associated with age-related cellloss, loss of mitochondrial function, loss of telomere function, anabnormal proliferative disease, a degenerative disease, or afunction-decreasing disorder.
 58. (canceled)
 59. The method of claim 50,wherein the age-related disease or disorder is selected fromtumorigenesis, malignant cancer development, myocardial infarction,cerebellar infarction, stroke, Parkinson's disease, heart failure,atherosclerosis, hypertension, cataract, age-related maculardegeneration, sarcopenia, osteoarthritis, osteoporosis, loss of bonemarrow, multiple sclerosis, Sjogren, Rheumatoid arthritis, decreasedimmune function, diabetes, idiopathic pulmonary fibrosis, aneurodegenerating disease, Alzheimer's disease, Huntington's disease,and disorders caused by dysfunction in testosterone, estrogen, growthhormone, IGF-I, or energy production.
 60. The method of claim 50,wherein the effective amount of the lithium compound is from about 1 mgto about 50 mg and the effective amount of the glycyrrhizic triterpenoidcompound is from about 1 to about 100 mg.
 61. (canceled).
 62. The methodof claim 50, further comprising administrating to the subject aneffective amount of at least one retinoic acid compound or apharmaceutically acceptable salt thereof. 63.-92. (canceled)
 93. Themethod of claim 1, wherein the effective amount of the lithium compoundis from about 1 mg to about 50 mg and the effective amount of theglycyrrhizic triterpenoid compound is from about 1 to about 100 mg.